Molecular detection systems utilizing reiterative oligonucleotide synthesis

ABSTRACT

The present invention provides methods for detecting the presence of a target molecule by generating multiple detectable oligonucleotides through reiterative enzymatic oligonucleotide synthesis events on a defined polynucleotide sequence. The methods generally comprise using a nucleoside, a mononucleotide, an oligonucleotide, or a polynucleotide, or analog thereof, to initiate synthesis of an oligonucleotide product that is substantially complementary to a target site on the defined polynucleotide sequence; optionally using nucleotides or nucleotide analogs as oligonucleotide chain elongators; using a chain terminator to terminate the polymerization reaction; and detecting multiple oligonucleotide products that have been synthesized by the polymerase. In one aspect, the invention provides a method for detecting a target protein, DNA or RNA by generating multiple detectable RNA oligoribonucleotides by abortive transcription.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/984,664, filed Oct. 30, 2001, now U.S. Pat. No. 7,045,319 B2, issuedMay 16, 2006; and incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the detection and kits forthe detection of target molecules and, more particularly, to nucleicacid-based detection assays that produce multiple signals from a targetmolecule by generating multiple copies of detectable oligonucleotidesthrough reiterative synthesis events on a defined nucleic acid template,particularly via abortive transcription initiation. The method and kitsof the invention may be used to detect mutations, RNA molecules,pathogens, proteins, or pre-cancerous conditions.

2. Related Art

The development of various methods for nucleic acid detection and thedetection of nucleic acid amplification products has led to advances inthe detection, identification, and quantification of nucleic acidsequences in recent years. Nucleic acid detection is potentially usefulfor both qualitative analyses, such as the detection of the presence ofdefined nucleic acid sequences, and quantitative analyses, such as thequantification of defined nucleic acid sequences. For example, nucleicacid detection may be used to detect and identify pathogens; detectgenetic and epigenetic alterations that are linked to definedphenotypes; diagnose genetic diseases or the genetic susceptibility to aparticular disease; assess gene expression during development, disease,and/or in response to defined stimuli, including drugs; as well asgenerally foster advancements in the art by providing researchscientists with additional means to study the molecular and biochemicalmechanisms that underpin cellular activity.

Nucleic acid detection technology generally permits the detection ofdefined nucleic acid sequences through probe hybridization, that is, thebase-pairing of one nucleic acid strand with a second strand of acomplementary, or nearly complementary, nucleic acid sequence to form astable, double-stranded hybrid. Such hybrids may be formed of aribonucleic acid (RNA) segment and a deoxyribonucleic acid (DNA)segment, two RNA segments, or two DNA segments, provided that the twosegments have complementary or nearly complementary nucleotidesequences. Under sufficiently stringent conditions, nucleic acidhybridization may be highly specific, requiring exact complementaritybetween the hybridized strands. Typically, nucleic acid hybrids comprisea hybridized region of about eight or more base pairs to ensure thebinding stability of the base-paired nucleic acid strands. Hybridizationtechnology permits the use of one nucleic acid segment, which isappropriately modified to enable detection, to “probe” for and detect asecond, complementary nucleic acid segment with both sensitivity andspecificity. In the basic nucleic acid hybridization assay, asingle-stranded target nucleic acid (either DNA or RNA) is hybridized,directly or indirectly, to a labeled nucleic acid probe, and theduplexes containing the label are quantified. Both radioactive andnon-radioactive labels have been used.

However, a recognized disadvantage associated with nucleic acid probetechnology is the lack of sensitivity of such assays when the targetsequence is present in low copy number or dilute concentration in a testsample. In many cases, the presence of only a minute quantity of atarget nucleic acid must be accurately detected from among myriad othernucleic acids that may be present in the sample. The sensitivity of adetection assay depends upon several factors: the ability of a probe tobind to a target molecule; the magnitude of the signal that is generatedby each hybridized probe; and the time period available for detection.

Several methods have been advanced as suitable means for detecting thepresence of low levels of a target nucleic acid in a test sample. Onecategory of such methods is generally referred to as targetamplification, which generates multiple copies of the target sequence,and these copies are then subject to further analysis, such as by gelelectrophoresis, for example. Other methods generate multiple productsfrom a hybridized probe, or probes, by, for example, cleaving thehybridized probe to form multiple products or ligating adjacent probesto form a unique, hybridization-dependent product. Still other methodsamplify signals generated by the hybridization event, such as a methodbased upon the hybridization of branched DNA probes that have a targetsequence binding domain and a labeled reporting sequence binding domain.

There are many variations of target nucleic acid amplification,including, for example, exponential amplification, ligation-basedamplification, and transcription-based amplification. An example of anexponential nucleic acid amplification method is the polymerase chainreaction (PCR), which has been disclosed in numerous publications. See,for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Mullis et al. U.S. Pat. No. 4,582,788; and Saiki R.et al. U.S. Pat. No. 4,683,194. An example of ligation-basedamplification is the ligation amplification reaction (LAR) which isdisclosed by Wu et al. in Genomics 4:560 (1989). Various methods fortranscription-based amplification are disclosed in U.S. Pat. Nos.5,766,849 and 5,654,142; and also in Kwoh et al., Proc. Natl. Acad. Sci.U.S.A. 86:1173 (1989).

The most commonly used target amplification method is the polymerasechain reaction (PCR), which consists of repeated cycles of DNApolymerase-generated primer extension reactions. Each reaction cycleincludes heat denaturation of the target nucleic acid; hybridization tothe target nucleic acid of two oligonucleotide primers, which bracketthe target sequence on opposite strands of the target that is to beamplified; and extension of the oligonucleotide primers by a nucleotidepolymerase to produce multiple, double-stranded copies of the targetsequence. Many variations of PCR have been described, and the method isbeing used for the amplification of DNA or RNA sequences, sequencing,mutation analysis, and others. Thermocycling-based methods that employ asingle primer have also been described. See, for example, U.S. Pat. Nos.5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. The primercan be a DNA/RNA chimeric primer, as disclosed in U.S. Pat. No.5,744,308. Other methods that are dependent on thermal cycling are theligase chain reaction (LCR) and the related repair chain reaction (RCR).

Target nucleic acid amplification may be carried out through multiplecycles of incubation at various temperatures (i.e., thermal cycling) orat a constant temperature (i.e., an isothermal process). The discoveryof thermostable nucleic acid modifying enzymes has contributed to rapidadvances in nucleic acid amplification technology. See, Saiki et al.,Science 239:487 (1988). Thermostable nucleic acid modifying enzymes,such as DNA and RNA polymerases, ligases, nucleases, and the like, areused in methods that are dependent on thermal cycling as well as inisothermal amplification methods.

Isothermal methods, such as strand displacement amplification (SDA) forexample, are disclosed by Fraiser et al. in U.S. Pat. No. 5,648,211;Cleuziat et al. in U.S. Pat. No. 5,824,517; and Walker et al., Proc.Natl. Acad. Sci. U.S.A. 89:392-396 (1992). Other isothermal targetamplification methods include transcription-based amplification methodsin which an RNA polymerase promoter sequence is incorporated into primerextension products at an early stage of the amplification (WO 89/01050),and a further, complementary, target sequence is amplified throughreverse transcription followed by physical separation or digestion of anRNA strand in a DNA/RNA hybrid intermediate product. See, for example,U.S. Pat. Nos. 5,169,766 and 4,786,600. Further examples oftranscription-based amplification methods include Transcription MediatedAmplification (TMA), Self-Sustained Sequence Replication (3SR), NucleicAcid Sequence Based Amplification (NASBA), and variations there of. See,for example, Guatelli et al. Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878(1990) (3SR); U.S. Pat. No. 5,766,849 (TMA); and U.S. Pat. No. 5,654,142(NASBA).

These and other techniques have been developed recently to meet thedemands for rapid and accurate detection of pathogens, such as bacteria,viruses, and fungi, for example, as well as the detection of normal andabnormal genes. While all of these techniques offer powerful tools forthe detection and identification of minute amounts of a target nucleicacid in a sample, they all suffer from various problems.

One problem, especially for PCR, is that conditions for amplifying thetarget nucleic acid for subsequent detection and optional quantitationvary with each test, that is, there are no constant conditions favoringtest standardization. Further, amplification methods that use athermocycling process have the added disadvantage of extended lag timeswhich are required for the thermocycling block to reach the “target”temperature for each cycle. Consequently, amplification reactionsperformed using thermocycling processes require a significant amount oftime to reach completion. The various isothermal target amplificationmethods do not require a thermocycler and are therefore easier to adaptto common instrumentation platforms. However, the previously describedisothermal target amplification methods also have several drawbacks.Amplification according to the SDA methods requires the presence ofdefined sites for restriction enzymes, which limits its applicability.The transcription-based amplification methods, such as the NASBA and TMAmethods, are limited by the need to incorporate a polymerase promotersequence into the amplification product by a primer.

Accordingly, there is a need for rapid, sensitive, and standardizednucleic acid signal detection methods that can detect low levels of atarget nucleic acid in a test sample. These needs, as well as others,are met by the inventions of this application.

All patents, patent publications, and scientific articles cited oridentified in this application are hereby incorporated by reference intheir entirety to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by referencein its entirety.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for producing multipledetectable signals through reiterative oligonucleotide synthesisreactions on a defined polynucleotide for the detection of targetmolecules. The invention also provides applications for the reiterativesynthesis and detection methods. Important applications of the methodsand kits of the invention, include but are not limited to detection ofmutations and single nucleotide polymorphisms, RNA molecules, pathogens,and detection of pre-cancerous or cancerous mutations and conditions.

Accordingly, in one aspect, the invention provides a method forsynthesizing multiple complementary oligonucleotides from a target DNAor RNA polynucleotide. The method comprises the steps of: (a)hybridizing an initiator (nucleoside, mononucleotide, oligonucleotide orpolynucleotide) with a single-stranded target polynucleotide (RNA orDNA); (b) incubating said target polynucleotide and initiator with anRNA-polymerase, a terminator, and optionally additional ribonucleotides;(c) synthesizing multiple oligonucleotides from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotides, thereby synthesizingmultiple reiterative oligonucleotides.

In another aspect, the invention provides a method for detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of (a) hybridizing aninitiator with a single stranded target polynucleotide; (b) incubatingsaid target polynucleotide and initiator with an RNA-polymerase, aterminator and optionally additional ribonucleotides; (c) synthesizingmultiple oligonucleotides from said target polynucleotide, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple reiterativeoligonucleotides; and (d) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts of a polynucleotide of interest.

In a further aspect, the invention provides a method of detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of: (a) hybridizing aninitiator to a single-stranded target polynucleotide; (b) incubatingsaid target polynucleotide and initiator with a target site probe, anRNA-polymerase, a terminator and optionally additional ribonucleotides,wherein said target site probe hybridizes with said targetpolynucleotide; (c) synthesizing an oligonucleotide transcript that iscomplementary to said target site from said target polynucleotide,wherein said initiator is extended until said terminator is incorporatedinto said oligonucleotide transcript, thereby synthesizing multiplereiterative oligonucleotide transcripts; and (d) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts,wherein said oligonucleotides being synthesized are one of the lengthsselected from the group consisting of: about 2 to about 26 nucleotides,about 26 to about 50 nucleotides and about 50 nucleotides to about 100nucleotides, and greater than 100 nucleotides.

In a further aspect, the invention provides a method for detectingmethylated cytosine residues at CpG sites in a target polynucleotide.The method comprises the steps of: (a) deaminating a single-strandedtarget DNA sequence under conditions which convert unmethylated cytosineresidues to uracil residues while not converting methylated cytosineresidues to uracil; (b) hybridizing an initiator with a single strandedtarget polynucleotide; (c) incubating said deaminated targetpolynucleotide and said initiator with a terminator, an RNA-polymeraseand optionally additional ribonucleotides, wherein at least one of saidinitiator, terminator, or optional ribonucleotides is modified to enabledetection of hybridization to the CG sites; (d) synthesizing anoligonucleotide transcript that is complementary to said CG sites fromsaid target polynucleotide, wherein said initiator is extended untilsaid terminator is incorporated into said oligonucleotide transcriptthereby synthesizing multiple reiterative oligonucleotide transcripts;and (e) detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts.

In still a further aspect, the invention provides a method for detectingmethylated cytosine residues at a CpG site in a target gene. The methodcomprises the steps of: (a) deaminating a single-stranded target DNApolynucleotide under conditions which convert unmethylated cytosineresidues to uracil residues while not converting methylated cytosineresidues to uracil; (b) hybridizing a target site probe with said singlestranded target polynucleotide; (c) incubating said targetpolynucleotide and target site probe with, an initiator, a terminator,an RNA-polymerase, and optionally additional ribonucleotides, whereinsaid at least one of said initiator, said terminator or said nucleotidesare complementary to the CpG site; (d) synthesizing an oligonucleotidetranscript that is complementary to said target site from said targetpolynucleotide, wherein said initiator is extended until said terminatoris incorporated into said oligonucleotides, thereby synthesizingmultiple reiterative oligonucleotide transcripts; and (e) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts.

In still a further aspect, the invention provides a method for detectingthe presence or absence of mutations in a target DNA sequence. Themethod comprises the steps of: (a) hybridizing a target site probe to asingle-stranded DNA polynucleotide, wherein said DNA polynucleotide maycontain a mutation relative to a normal or wild type gene; (b)incubating said target polynucleotide and target-site probe with anRNA-polymerase, a initiator, a terminator and optionally additionalribonucleotides; (c) synthesizing an oligonucleotide transcript fromsaid target polynucleotide that is complementary to a target mutationsite, wherein said initiator is extended until said terminator isincorporated into said oligonucleotides thereby synthesizing multipleabortive reiterative oligonucleotides; and (d) determining the presenceor absence of a mutation by detecting or quantifying said reiterativelysynthesized oligonucleotides transcribed from said target DNApolynucleotide.

In another aspect, the invention provides a method for detectingmutations in a target DNA polynucleotide using a capture probe. Themethod comprises the steps of: (a) immobilizing a capture probe designedto hybridize with said target DNA polynucleotide; (b hybridizing saidcapture probe to said target DNA polynucleotide, wherein said DNApolynucleotide may contain a mutation relative to a normal or wild typegene; (c) incubating said target polynucleotide and with anRNA-polymerase, initiator, a terminator and optionally additionalribonucleotides; (d) synthesizing an oligonucleotide transcript that iscomplementary to a target site from said target polynucleotide, whereinsaid initiator is extended until said terminator is incorporated intosaid oligonucleotide transcript, thereby synthesizing multiple abortivereiterative oligonucleotide transcripts; and (e) determining thepresence or absence of a mutation by detecting or quantifying saidreiteratively synthesized oligonucleotide transcripts from said targetDNA polynucleotide.

In another aspect, the invention provides a method for detecting DNA orRNA in a test sample. The method comprises the steps of: (a) hybridizinga single stranded target polynucleotide with an abortive promotercassette comprising a sequence that hybridizes to the single strandedtarget polynucleotide, and a region that can be detected bytranscription by a polymerase; (b) incubating said target polynucleotidewith an RNA-polymerase, an initiator, a terminator and optionallyadditional ribonucleotides; (c) synthesizing an oligonucleotidetranscript that is complementary to the initiation start site of theAPC, wherein said initiator is extended until said terminator isincorporated into said oligonucleotides, thereby synthesizing multiplereiterative oligonucleotide transcripts; and (d) detecting orquantifying said reiteratively synthesized oligonucleotide transcripts.

In another aspect, the invention provides a method for detecting thepresence of pathogens in a test sample. The method comprises the stepsof: (a) hybridizing a single stranded target pathogen polynucleotide insaid test sample with an abortive promoter cassette comprising a regionthat can be detected by transcription by a polymerase; (b) incubatingsaid target polynucleotide and initiator with an RNA-polymerase, aterminator and optionally additional ribonucleotides; (c) synthesizingan oligonucleotide transcript that is complementary to initiation startsite of the APC, wherein said initiator is extended until saidterminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (d) determining the presence of a pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said test sample.

In still a further aspect, the invention provides a method for detectingpathogens in a test sample using a capture probe. The method comprisesthe steps of: (a) immobilizing a capture probe designed to hybridizewith a target DNA polynucleotide in said test sample; (b) hybridizingsaid capture probe with a test sample that potentially contains saidtarget DNA polynucleotide; (c) hybridizing a single stranded target DNApolynucleotide in said test sample with an abortive promoter cassettecomprising a region that hybridizes to the single stranded targetpathogen polynucleotide, and a region that can be detected bytranscription by a polymerase; (d) incubating said target polynucleotidewith an RNA-polymerase, initiator, a terminator and optionallyadditional ribonucleotides; (e) synthesizing an oligonucleotidetranscript that is complementary to said initiation transcription startsite of APC, wherein said initiator is extended until said terminator isincorporated into said oligonucleotides thereby synthesizing multiplereiterative oligonucleotide transcripts; and (f) determining thepresence or absence of a pathogen by detecting or quantifying saidreiteratively synthesized oligonucleotide transcripts.

In still a further aspect, the invention provides a method for detectingmRNA expression in a test sample. The method comprises the steps of: (a)hybridizing a target mRNA sequence with an abortive promoter cassettecomprising a region that can be detected by transcription by apolymerase; (b) incubating said target mRNA sequence with anRNA-polymerase, an initiator, a terminator and optionally additionalribonucleotides; (c) synthesizing an oligonucleotide transcript that iscomplementary to transcription initiation start site, wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotide transcript, thereby synthesizing multiple reiterativeoligonucleotides; and (d) determining the presence or absence of themRNA by detecting or quantifying said reiteratively synthesizedoligonucleotide transcripts synthesized from said test sample.

In still a further aspect, the invention provides a method for detectingan oligonucleotide synthesized from a target DNA sequence. The methodcomprises the steps of: (a) hybridizing a DNA primer with asingle-stranded target DNA sequence; (b) extending said DNA primer witha DNA polymerase and nucleotides, such that said DNA polymerasereiteratively synthesizes a nucleotide sequence; and (c) detectingoligonucleotide comprised of repeat sequences synthesized by said DNApolymerase.

In still a further aspect, the invention provides a method for producinga microarray. The method comprises the steps of: (a) synthesizingmultiple abortive oligonucleotide replicates from a target DNA sequenceby the method of claim 1; and (b) attaching said multiple abortiveoligonucleotide replicates to a solid substrate to produce a microarrayof said multiple abortive oligonucleotide replicates.

In still a further aspect, the invention provides a method for detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide. The method comprises the steps of: (a) incubating asingle-stranded target polynucleotide in a mixture comprising aninitiator, an RNA-polymerase and optionally additional ribonucleotides;(b) synthesizing multiple oligonucleotide transcripts from said targetpolynucleotide, wherein said initiator is extended until terminated dueto nucleotide deprivation, thereby synthesizing multiple abortivereiterative oligonucleotide transcripts; and (c) detecting orquantifying said reiteratively synthesized oligonucleotides.

In still a further aspect, the invention provides a method of detectingmultiple reiterated oligonucleotides from a target DNA or RNApolynucleotide with a target site probe. The method comprises the stepsof: (a) incubating a single-stranded target polynucleotide in a mixturecomprising an initiator, an RNA-polymerase, a target site probe andoptionally additional ribonucleotides, wherein said target site probeand said target polynucleotide hybridize to form a bubble complexcomprising a first double-stranded region upstream of a target site, asingle-stranded region comprising said target site, and a seconddouble-stranded region downstream of said target site; (b) synthesizingmultiple oligonucleotide transcripts from said target polynucleotide,wherein said initiator is extended until terminated due to nucleotidedeprivation, thereby synthesizing multiple abortive reiterativeoligonucleotides; and (c) detecting or quantifying said reiterativelysynthesized oligonucleotide transcripts.

In still a further aspect, the invention provides a method for detectingmethylated cytosine residues at a CG site near a target gene. The methodcomprises the steps of:

(a) deaminating a single-stranded target DNA sequence under conditionswhich convert unmethylated cytosine residues to uracil residues whilenot converting methylated cytosine residues to uracil; (b) incubating asingle-stranded target polynucleotide in a mixture comprising aninitiator, a terminator, an RNA-polymerase, a target site probe andoptionally additional ribonucleotides; (c) synthesizing multipleoligonucleotide transcripts from said target polynucleotide, whereinsaid initiator is extended until terminated due to nucleotidedeprivation, thereby synthesizing multiple abortive reiterativeoligonucleotide transcripts; and (d) detecting or quantifying saidreiteratively synthesized oligonucleotides.

In still a further aspect, the invention provides a method for detectinga target protein in a test sample. The method comprises the steps of:(a) covalently attaching the target protein to an abortive promotercassette (APC) by a reactive APC linker, wherein said APC comprises aregion that can be detected by transcription by a polymerase; (b)incubating said target protein with an RNA-polymerase, an initiator, aterminator and optionally additional ribonucleotides; (c) synthesizingan oligonucleotide transcript that is complementary to transcriptioninitiation start site of APC, wherein said initiator is extended untilsaid terminator is incorporated into said oligonucleotide transcript,thereby synthesizing multiple reiterative oligonucleotide transcripts;and (d) determining the presence or absence of the target protein bydetecting or quantifying said reiteratively synthesized oligonucleotidetranscripts synthesized from said test sample.

In still a further aspect, the invention provides a method for detectingcancer. The method comprises the steps of: (a) obtaining a tissue samplefrom a patient in need of detection of a cancer; (b) deaminating the DNAunder conditions which convert unmethylated cytosine residues to uracilresidues while leaving the methylated cytosine residues unaltered; (c)hybridizing an initiator to a target polynucleotide wherein saidinitiator is a mononucleoside, mononucleotide, binucleotide,oligonucleotide, polynucleotide, or an analog thereof; (d) incubatingsaid deaminated target polynucleotide and said initiator with aterminator, an RNA-polymerase and optionally additional ribonucleotides,wherein at least one of said initiator, terminator, or optionalribonucleotides is modified to enable detection of hybridization to theCG sites; (e) synthesizing an oligonucleotide transcript that iscomplementary to said CG sites from said target polynucleotide, whereinsaid initiator is extended until said terminator is incorporated intosaid oligonucleotide transcript thereby synthesizing multiplereiterative oligonucleotide transcripts; (f) detecting or quantifyingsaid reiteratively synthesized oligonucleotide transcripts; and (g)comparing the results with those obtained similarly from a controlsample.

In still a further aspect, the invention provides a method for detectingpathogens. The method comprises the steps of: (a) obtaining a sample inneed of detection of a pathogen; (b) hybridizing a single strandedtarget pathogen polynucleotide in said sample with an abortive promotercassette comprising a nucleotide sequence that hybridizes to singlestranded target pathogen polynucleotide, and a region that can bedetected by transcription by a polymerase; (c) incubating said targetpolynucleotide and initiator with an RNA-polymerase, a terminator andoptionally additional ribonucleotides; (d) synthesizing anoligonucleotide transcript that is complementary to initiation startsite of the APC, wherein said initiator is extended until saidterminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (e) determining the presence of a pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized from said test sample.

The present invention also provides kits for conducting theoligonucleotide synthesis and detection methods described herein. In oneaspect, for example, the invention provides reagent containers, whichcontain various combinations of the components described herein. Thesekits, in suitable packaging and generally (but not necessarily)containing suitable instructions, contain one or more components used inthe oligonucleotide synthesis and detection methods. The kit may alsocontain one or more of the following items: polymerization enzymes,initiators, primers, buffers, nucleotides, control DNA, antibodies,streptavidin, and biotin. The kit may also contain reagents mixed inappropriate amounts for performing the methods of the invention. Thereagent containers preferably contain reagents in unit quantities thatobviate measuring steps when performing the subject methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Abortive Promoter Cassettes. Abortive Promoter Cassettes (APC)are regions of nucleic acid that form a polymerase binding site and canbe attached to other macromolecules through interaction with a specificnucleic acid sequence, which is termed the APC linker. APC linkers canbe attached to target nucleic acids (DNA or RNA) by hybridization tocomplementary sequences on either the template or non-template strandsof the target nucleic acid. An APC Linker can also hybridize to acomplementary sequence placed on any target molecule, such as a protein,for detection of molecules that bind to said protein. Multipledetectable oligonucleotides are generated by polymerase bound to theAbortive Promoter Cassette. In this figure, the APC depicted containstwo regions of essential complementarity (A, A′ and C, C′), which areseparated by a “bubble region.” In this schematic, the “bubble region”is generated because regions of the two strands are non-complementary(B, and E). Alternatively, the APC may have two completely complementarystrands. Upon binding of the RNA polymerase, the DNA strands separate,which leads to the formation of the “bubble region.”

Regions A, B, and C are on one strand. Regions C′, E, and A′ are on thecomplementary strand. The APC may be made from two separate strands (ABCand C′EA′) or all 6 regions may be on a single polynucleotide, in whichregions C and C′ are separated by a linker region D, which can modifiedto be as long as needed. Linker region D may serve only to join C and C′or the sequence of region D may serve as a binding site for otherfactors that may enhance abortive transcription, such as transcriptionroadblock proteins, including but not limited to EcoRI QIII mutant, thelac repressor and other RNA polymerases. The linker region D may bedesigned for a single road block protein, or multiple roadblockproteins. The length of linker region D will depend on the function ofthe linker region.

FIG. 2: Signal Generation by Reiterative oligonucleotide synthesis. Asignal is generated by the enzymatic incorporation of one or morenucleotide analogs into multiple (n) highly similar or identicaloligonucleotide products. Under appropriate conditions, RNAoligonucleotides can be made from nucleic acid templates in the absenceof a promoter. An initiator may be comprised of one or more nucleosides,nucleoside analogs, nucleotides, or nucleotide analogs. The initiar maycontain one or more covalently joined nucleotides, including but notlimited to, 1-25 nucleotides, 26-50 nucleotides, 51-75 nucleotides,76-100 nucleotides, 101-125 nucleotides, and 126-150 nucleotides,151-175 nucleotides, 176-200 nucleotides, 201-225 nucleotides, 226-250nucleotides and more than 250 nucleotides, and may contain a functionalR group. The initiator (n copies) can be elongated directly with ncopies of a terminator to end chain elongation or n copies of otherelongator nucleotides (Y positions) may be incorporated between theinitiator and the terminator to form a longer oligonucleotide. Theterminator may contain a second functional group. N_(I)=Initiatingmononucleotide or oligonucleotide analog, N_(E)=Elongatingmononucleotides or analog, N_(T)=Terminating mononucleotide or analog,Z=x+y; R₁=H, OH, or reporter group; R₂=H, OH, or reporter group; N=deoxyor ribonucleotides; Polymerase=RNA-dependent or DNA-dependent RNApolymerase. DNA or RNA may be attached to other molecules, such asproteins

FIG. 3: 5′ AEDANS-S-AMP synthesis. Example of a mononucleotidetranscription initiator: IAEDANS (5-((2-((iodoacetyl)amino)ethyl)amino-1-Napthalenesulfonic acid alkylates AMPS(5-α-thio-AMP) to form the fluorescent transcription initiator. Thisanalog can only initiate transcription because it lacks a 5′triphosphate group and can therefore not be incorporated internally orterminally.

FIG. 4: Nucleotides that can be elongators or terminators. Nucleotideanalogs that may be included at internal or 3′ terminal positions inoligonucleotides are shown. All of these analogs can be converted toterminators simply by replacement of the 3′ OH group.

FIG. 5: Other fluorescent groups that may be R₁ or R₂. Theoligonucleotides can be labeled with a variety of functional groups.Several of the preferred fluorescent groups are shown.

FIG. 6: Dinucleotide synthesis via abortive initiation onsingle-stranded DNA or RNA. Single stranded (ss) nucleic acid is DNA orRNA. Polymerase is a DNA-dependent or RNA-dependent RNA polymerase.N_(I)=3′-OH nucleoside or nucleotide initiator; N_(T)=5′-triphosphatenucleotide or nucleotide analog terminator. R₁ may be on the 5′phosphate group, the 2′ position of the sugar, or on the purine orpyrimidine base. R₂ may be on the pyrimidine or purine base or 2′ or 3′position of the sugar/ribose or deoxyribose. R₁═H, OH, and/or anyreporter group or reporter group precursor, as described herein. R₂═H,OH, and/or any reporter group or reporter group precursor, as describedherein. Signal may be any signal that can be detected, and includes butis not limited to fluorescence, fluorescence resonance energy transfer(FRET), or colorimetric. As one example, R₁ may be AEDANS, and R₂ may beFluorescein. Signal is generated by FRET from R₁ to R₂.

FIG. 7: 5′-AEDANS-S_(P)A_(P)U-FLUORESCEIN. Dinucleotide generated byabortive initiation for FRET detection. When excited by light of theappropriate wavelength, R₁ (AEDANS) donates fluorescent energy to R₂(fluorescein), which then emits fluorescent light of a differentwavelength that can be detected and quantified. This fluorescenceresonance energy transfer (FRET) only occurs when the two groups arejoined together to form AEDANS-SpApU-Fluorescein during transcription,which brings the two groups close enough to each other for efficientenergy transfer.

FIG. 8: Signal generation via dinucleotide production. Oligonucleotidescan be synthesized that contain one R group on the initiator nucleotideand another on the terminator nucleotide, such that the R groups havedifferent functions. For example, if R₁ is biotin, it can be used foroligonucleotide product immobilization and R₂ allows for signalproduction.

Example 1: R₁ tag=biotin, R₂ tag=fluorescein: detection of fluoresceinemission

Example 2: R₁ tag=DNP, R₂ tag=reactive thiol

FIG. 9: Signal generation for FRET detection by abortive initiation.Oligonucleotides can be reiteratively synthesized that contain 2 to 25nucleotides and have two different R groups, one at or near each end ofthe oligonucleotide product made during transcription. Energy transferbetween the two R groups on the substrates can only occur after they arebrought into proximity during template-directed oligonucleotidesynthesis by enzymatic phosphodiester bond formation between the labeledinitiator and the labeled terminator nucleotides. The R₁ donor group onN_(I) can be excited by irradiating the sample with light of wavelengthof λ_(1A), where λ_(1A) is the absorption maximum of group R₁. Theexcited R₁ donor group emits light of wavelength λ_(1E), where λ_(1E) isthe emission maximum for group R₁ and also a wavelength for absorptionby group R₂ (λ_(2A)). The acceptor R₂ group on N_(T) absorbs light ofwavelength λ_(1E)/λ_(2A) that was emitted by the excited R₁ donor groupon N_(I). The excited acceptor R₂ group on N_(T) emits light ofwavelength λ_(2E), which is detected and quantified. Similarly, R₂ maybe an energy donor to R₁, with emission from R₁ detected. In the absenceof target-associated template, no oligonucleotide is synthesized.

FIG. 10: Trinucleotide energy transfer. Labeled oligonucleotidesynthesis is initiated with a labeled dinucleotide initiator. The labelmay be on either the 5′ nucleotide (R₁) or the 3′ nucleotide (R₂) of thedinucleotide initiator. The initiator is elongated with a labeled (R₃)5′-nucleosidetriphosphate terminator nucleotide analog. Detection viaenergy transfer can be adjusted to utilize R₁ or R₂ with R₃, as shown.In the absence of nucleic acid template-directed phosphodiester bondformation between the initiator and terminator, the R groups remainsufficiently separated that no energy transfer is detected. In thisexample, the amount of energy emitted as λ_(3E) is directly proportionalto the amount of template-associated target present. Similarly, the Rgroups may be varied for other applications, as demonstrated in FIG. 8.

FIG. 11: Target Site Probe. An RNA polymerase can be directed tospecific nucleotide positions (sites) in target nucleic acids by thehybridization of a gene-specific or region-specific Target Site Probe(TSP). The target site is a nucleotide position in the DNA to beanalyzed for sequence (as in detection of single nucleotidepolymorphisms) or structure (as in assessing the methylation status of aspecific nucleotide), and it is located on the template strand of thetarget sequence at the junction of regions E and C′ in the targetsequence. The TSP contains a region of homology to the target nucleicacid (Region A) which begins approximately 8-14 nucleotides and endsapproximately 15-35 nucleotides upstream of the target site nucleotide.A second region of the TSP is designed to be non-complementary to the8-14 nucleotides immediately upstream of the target site (Region B), sothat a melted “bubble” region forms when the TSP hybridizes to thetarget nucleic acid. The TSP contains a third region (Region C) which isessentially complementary to the 5-25 nucleotides immediately downstreamof the target site nucleotide. RNA polymerase will bind to the bubblecomplex such that transcription will start at the E/C′ junction and willmove downstream into the C/C′ hybrid.

FIG. 12: Methylation of CpG Islands in DNA. The human genome has a 4-5fold lower frequency of CpG dinucleotides than expected given theoverall frequency of C and G in human DNA. A large fraction of CpGsequence is distributed into clusters known as CpG islands. Thesesequence patterns are between 300-3000 nucleotides long and overlap withabout 60% of all human promoters. The remaining CpG dinucleotidesoutside of CpG islands contain methylated C. CpG methylation outside ofCpG islands stabilize the genome by inactivating the expression ofparasitic DNA, and independently play an essential role in development.Changes in the methylation status of cytosine in CpG islands are earlyevents in many cancers and permanent changes found in many tumors. TheseCpG islands are found in the regions next to genes that determinewhether the gene is “ON” or “OFF”. Many genes that are important forpreventing cancer, such as tumor suppressor genes, need to be “ON” forcells to grow normally. Cellular enzymes can add methyl groups(methylation) to the C residues in these CpG islands. This methylationresults in the shutting “OFF” of these genes. When tumor suppressorgenes are shut “OFF”, the cell no longer makes the proteins that theyencode, and the cell begins to grow without control checkpoints. This isone of the early events that can lead to cell “transformation” and theprogression of cancer.

FIG. 13: Deamination conversion of unmethylated cytosine groups in DNA.Deamination converts unmethylated C to U. Methylated C groups, such asthose in CpG islands that regulate eukaryotic genes, are resistant todeamination and remain as C in the product DNA. If 100% deaminationoccurs, methylated DNA will still contain CpG doublets, whereasunmethylated DNA will contain no cytosine and will now contain UpG whereCpG doublets were before deamination. This difference in DNA sequencecan be used to distinguish between methylated and unmethylated DNA byabortive transcription because the two DNAs encode differentdinucleotides. (SEQ ID NOS: 1, 2, 3 and 17).

FIG. 14: Detection of methylation using dinucleotide synthesis.Dinucleotide synthesis can be used to assess the overall methylationstate of DNA. In the presence of RNA polymerase, CTP or a CTP analog(R1-C—OH), and GTP or a GTP analog (R1-CpG-R2), the deaminatedmethylated DNA template will produce n copies of a labeled dinucleotideproduct, where n is proportional to the number of methylated CpGdinucleotides in the starting DNA. The deaminated unmethylated DNAtemplate can produce no dinucleotide with these substrates because thetemplate no longer encodes “C” at any position. (SEQ ID NOS: 4 and 5).

If R₁ and R₂ are appropriately labeled, the dinucleotide will produce asignal that is proportional to the number of methylated CpG sites. Forexample, if R₁ is a fluorescent energy donor or acceptor that iscompatible with a second donor or acceptor, R₂, a signal will bedetected by fluorescent resonance energy transfer (FRET) between R₁ andR₂ only when the two groups are brought into proximity afterincorporation into the dinucleotide in an enzymatic, template-dependentreaction. The reiterative synthesis of these dinucleotides duringabortive transcription results in multiple signals from each CpG targetand can be used to assess the methylation level of the DNA.

Similarly, abortive synthesis of trinucleotides by transcriptioninitiation with labeled dinucleotides that end in C (ApC, CpC, GpC, UpC)and termination with labeled GTP can be used to produce signal from thedeaminated methylated template, but not the deaminated unmethylatedtemplate. This trinucleotide synthesis approach may be expanded by theaddition of a site-specific oligonucleotide to allow assessment of themethylation status of a specific CpG site, rather than the entireisland, as illustrated in FIG. 15.

FIG. 15: Assessing methylation status of specific CpG sites in CpGislands by abortive initiation. Target site probes can be used toexamine the methylation status of specific CpG islands in specificgenes. In the deaminated methylated DNA, the dinucleotide CpG is encodedby the template at the 3 methylated sites 1, 3 and 4, but not by theunmethylated site 2. To specifically determine if Site 3 is methylatedand if so, to what extent, position (C21) can be targeted with a TargetSite Probe, as described in FIG. 11. The template C in question ispositioned at the junction of the bubble region and the downstreamduplex so that it encodes the next incorporated nucleotide forappropriately primed RNA polymerase that binds to the bubble region. Ifa labeled initiator R₁—N_(x)pC—OH is used, where N_(x) may be C for adinucleotide CpC initiator or N_(x) may be CpC for a trinucleotideinitiator, etc., the initiator can be elongated with a labeled GTPanalog pppG-R_(2G) to form a trinucleotide R₁N_(x)CpG-R₂. Similarly, ifthe C in question was not methylated, the position will now be a U andwill encode nucleotide A. If an ATP analog pppA-R_(2A) is present, itwill be incorporated opposite positions where the C was not methylated.If the GTP analog is labeled with group R_(2G), which is an energyacceptor from the R group on the initiator, R₁, then the amount ofR₁N_(x)CpGR_(2G), which will be proportional to the amount of methylatedC present at that position, can be quantified by measuring the emissionfrom R_(2G) at wavelength λ_(2GE). The similar situation exists forincorporation of the ATP analog and measurement of the emission from itsR group, also an energy acceptor from the initiator R₁. By determiningthe ratio of the magnitude of emission from the GTP analog to the totalemission from both the ATP and GTP analogs, the site can be assigned amethylation index M. If all of the Cs at that position are methylated,M=1. If none of the site is methylated, M=0. (SEQ ID NOS: 6, 7 and 8).

FIG. 16: Genes with altered methylation in cancer. Forty-nine genes withmethylation changes associated with cancer initiation and progressionare plotted versus 13 cancers. An oval indicates an abnormal methylationstatus for a gene, coded by cancer type. Cancer is actively preventedthrough the expression of close to 100 tumor-suppressor genes thatregulate the cell-division cycle. CpG methylation potentially is apowerful biomarker for cancer detection. Examination of the promoters oftumor suppressor genes from tumor biopsies suggests that CpG methylationis common enough to equal the impact of mutagenesis in tumor promotion.At least 60 tumor suppressor and repair genes are associated withabnormally high levels of CpG methylation across virtually all of thecommon tumor types. In virtually all cases, defective expression oftumor suppressor genes begins at an early stage in tumor progression.Detection of these early methylation events before advanced symptomsappear should improve the chances that a cancer will be treated while itis highly curable. CpG methylation patterns are frequently biased toparticular genes in particular types of cancers. Therefore, it should bepossible to develop methylation signatures for common cancers,indicating both cancer type and stage. Data on the methylation status ofmultiple promoters could give clues as to the location of a tumor incases where several organs can contribute to a sample. For example, shedbladder, kidney or prostate cells can populate a urine sample. Tumorsfrom each of these tissues are frequently associated with distinctcombinations of CpG island methylation.

FIG. 17: Single nucleotide polymorphism detection by abortiveoligonucleotide synthesis. The identity of a nucleotide at a specificposition can also be determined by abortive initiation in the presenceof target nucleic acid and a position-specific Target Site Probe. Thiscan be applied to SNP identification by initiating transcription with anoligonucleotide complementary to the DNA upstream from the SNP site. Forexample for synthesis of a trinucleotide, the dinucleotide initiatorwould be complementary to the know nucleotides at positions-1 and -2,relative to the SNP site.

FIG. 18: Detection and identification of single nucleotide polymorphisms(SNPs) by abortive transcription. The identity of a specific DNAnucleotide (A,C,G,T/dU) can be identified by abortive transcription withthe use of a Target Site Probe (TSP). For example, to determine whethera DNA contains a normal nucleotide (wild type) or a mutant nucleotide(point-mutation, single nucleotide polymorphism/SNP), a gene-specificTSP can be added to target DNA (or amplification/replication product)such that the SNP position corresponds to the last nucleotide in theC/C′ hybrid at the junction of the downstream duplex and the bubbleregion. A labeled initiator oligonucleotide (R₁NI-OH) that iscomplementary to the region upstream of the SNP site can be elongated byan RNA polymerase to add the next encoded nucleotide, corresponding tothe SNP. The labeled terminators (pppN_(T)—R₂ or pppU—R_(2U),pppA-R_(2A), pppC—R_(2C), pppG-R_(2G)) can each be labeled withdifferent R groups, for example, R_(2A), R_(2C), R_(2G) and R_(2U) couldeach be resonance energy acceptors from R₁, with each emitting lightwith a different detectable wavelength.

FIG. 19: Signal Generation from abortive promoter. An Abortive PromoterCassette (APC) consists of one or more oligonucleotides orpolynucleotides that together create a specific binding site for an RNApolymerase coupled to a linker region (APC linker) for attachment totarget molecules (DNA, RNA, Protein). The APC may contain an artificialpromoter, or it may contain the promoter for a specific RNA polymerase.For example, trinucleotide or tetranucleotide products that could begenerated from with a common phage RNA polymerase can be made with alabeled GpA or GpApA initiator and a labeled pppG or pppA terminator.(SEQ ID NOS: 9 and 10).

FIG. 20: Detection of nucleic acids by abortive transcription. Fordetection of nucleic acids, such as DNA or RNA associated with specificdiseases or with viral and bacterial pathogens, one can either detectthe nucleic acid directly or after replication or primer extension. Inthe first case, the APC linker in the Abortive Promoter Cassette wouldbe designed to be complementary to a known DNA or RNA sequence of thetarget nucleic acid. Alternatively, one or more copies of the target DNAor cDNA copies of target RNA can be made by primer extension or reversetranscription initiated with primers containing a universal APC linkersequence at the 5′ end. In either case, the target DNA or RNA can beretrieved from the sample by attachment to a solid support, for example,to which an oligonucleotide that contains a second target-specificsequence, which is termed a “capture sequence,” has been attached viaany number of immobilization tags, including but not limited to biotin,hexahistidine or any other hapten. Once attached, abortive transcriptionis initiated by addition of a polymerase and the appropriate labelednucleotides, which results in signal generation, as previouslydescribed.

FIG. 21: Detection of mRNA by Abortive Transcription. An AbortivePromoter Cassette for detection of mRNA will contain as its APC linkeran oligo T tail. This tail is complementary to the poly A tail found atthe 3′ end of eukaryotic mRNAs and will be used for attachment of theAPC to the target mRNA. The target mRNA can be retrieved from a sampleby attachment to an immobilized capture probe containing a capturesequence, which is complementary to some region of the target mRNA.

FIG. 22: Detection of proteins or other haptens/antigens with abortivetranscription. Signal generation via abortive initiation from anAbortive Promoter Cassette can be used to detect other molecules, suchas proteins. For example, an APC linker sequence can be prepared towhich thiol-reactive or amine-reactive protein crosslinking agents Rwill be covalently attached. The reactive APC linker will be added tothe target protein, which may be purified or in a complex mixture (suchas a cell lysate), and the APC linker will be covalently attached to thetarget protein via modification of protein thiol and/or amine groups.The labeled protein can then be immobilized utilizing a target-specificprobe (such as an antibody). The Abortive Promoter Cassette is thenattached via the APC linker, and signal is generated, as previouslydescribed.

FIG. 23: Enhanced detection of molecular targets via abortivetranscription on APC particles. Even greater detection sensitivity canbe achieved with the use of particles to which multiple copies,including tens, hundreds, thousands, tens of thousands or even more ofthe Abortive Promoter Cassette (APC) have been attached. The sphere willalso contain a linker that will be specific for binding to a group thatcan be attached to the target molecule. For example, streptavidin (SA)can be attached to the APC particles and biotin to the target molecule,which can then be immobilized via interaction with a target-specificcapture probe. Once the APC particles interact with the target, forexample via the SA-biotin interaction, polymerase and labelednucleotides can be added for signal generation, as described.

FIG. 24: Coating of DNA or RNA targets with APC particles forultra-sensitive detection or molecular imaging. An alternative methodfor the ultra-sensitive detection or visualization of target DNA or RNAcan be achieved by reverse transcription of target RNA or copying(single copy or amplification) of target DNA in the presence of probelabeled dNTP analogs. As an example. 5-SH-dUTP can be incorporated atvery high frequency in DNA molecules, which can then be immobilized andfurther modified with other groups, such as biotin. To this, APCparticles can be added, as described in FIG. 23, each of which willinteract with a nucleotide analog on the target. This essentially coatsthe target DNA or RNA with APC particles capable of generating multipleoligonucleotide products for a variety of methods of moleculardetection.

FIG 25. Detection of telomerase activity with reiterativeoligonucleotide synthesis. Reiterative oligonucleotide synthesis withDNA polymerases can also be used for signal generation, however, theproduct oligonucleotides need not be released, but may be joinedtandemly in the product. As an example, telomerase activity can bedetected by immobilizing a telomerase-specific probe to a solid matrixto capture cellular telomerase, which carries its own RNA template forDNA synthesis. For example, with human telomerase, the RNA template onthe enzyme encodes the DNA sequence GGGTTA. The capture probe maycontain the sequence GGGTTA, which will be added reiteratively to theend of the telomerase capture probe, if telomerase is present in thesample. Signal generation can be achieved in several ways, one of whichinvolves including one or more reporter tagged dNTPs in the synthesisreaction to produce a product that has multiple R₁ groups attached alongthe backbone of the DNA product. For detection, this product can then behybridized to a complementary probe containing nucleotides with a secondR group (R₂) attached that will hybridize to the R₁ labeled product.This will bring the R₁ and R₂ groups together for signal generation viaFRET from between R₁ and R₂, or via other methods. Alternatively,telomerase may incorporate 2 labeled nucleotides in the product DNA andlook for energy transfer between the 2 labeled nucleotides in the singlestrand of DNA. (SEQ ID NOS: 11, 12, 13 and 14).

FIG. 26. Synthesis of a dye labeled initiator. 5′EADANS-S-CMP wassynthesized from the conjugation of IAEDANS and α-S-CMP. The scannedimage of the thin layer chromatography plate shows the control IAEDANSand the IAEDANS labeled product. Lane 1:Cytidine-5′-O-(1-Thiomonophosphate); Lane 2:Cytidine-5′-O-(1-Thiotriphosphate); Lane 3:5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid(1,5-IAEDANS); Lane 4: Cytidine-5′-O-(1-Thiotriphosphate) and(1,5-IAEDANS); Lane 5: Cytidine-5′-O-(1-Thiomonophosphate) and(1,5-IAEDANS); Lane 6: Adenosine-5′-O-(1-Thiomonophosphate); Lane 7:Adenosine-5′-O-(1-Thiomonophosphate) and (1,5-IAEDANS); Lane 8:(1,5-IAEDANS); Lane 9: Cytidine-5′-O-(1-Thiotriphosphate); Lane 10:Cytidine-5′-O-(1-Thiomonophosphate). Lanes 4, 5, and 7 also contain 1 Uof E. coli RNA polymerase, Buffer T and 150 ng of denatured pBR322

FIG. 27. Abortive Transcription Initiation with labeled initiators. Thephotograph of the gel shows the results of an abortive transcriptioninitiation reaction using three different dinucleotide initiators, whichwere (1) ApG; (2) Biotin-ApG; and (3) 5′ TAMRA-SpApG, and a terminatingnucleotide, which was α³²P-UTP. All three dinucleotides allowed forincorporation of UTP in the 3^(rd) position to generate 5′TAMARA-SpApGpU. The unlabeled ApG incorporates more efficiently thandoes the Biotin-ApG, which incorporates more efficiently than theTAMARA-ApG.

FIG. 28. Abortive Transcription Initiation with a labeled terminator.The scanned image of the thin layer chromatography plate shows theresults of an abortive transcription initiation reaction using anunlabeled dinucleotide initiator, ApG, and a labeled terminator, whichwas 5′-SF-UTP (5-thioacetemidofluorescein-uridine 5′-triphosphate. Thelabeled terminator was efficiently incorporated to generate theoligonucleotide product ApGpU.

FIG. 29. Portion of the contig sequence of the CDKN2A gene. The sequencerepresents a small portion of the contig starting at 856630 nucleotidesfrom the start of the contig sequence. The sequence represents a CpGisland. Contig number: NT_(—)008410.4. (SEQ ID NO: 15).

FIG. 30. Schematic representation of a “capture probe” to determine themethylation status of a specific gene. Oligonucleotide probes that arespecific for a region near the CpG island of the target gene areimmobilized onto a microtiter plate. The DNA of interest is added to theimmobilized probe and bound to the capture probe. The DNA is thenchemically modified to convert unmethylated C to T, and leave methyl-Cunaffected. The converted DNA can then be amplified by an optional PCRstep to further enhance the signal. A labeled CpG initiator is thenadded with an RNA polymerase and labeled nucleotide(s).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and kits for detecting the presence of atarget molecule (such as nucleic acid sequence or protein) by generatingmultiple detectable oligonucleotides through reiterative synthesisevents on a defined nucleic acid. The methods generally comprise using alabeled nucleotide or oligonucleotide transcription initiator toinitiate synthesis of an abortive oligonucleotide product that issubstantially complementary to a defined site on a target nucleic acid;using a chain terminator to terminate the polymerization reaction; and,optionally, using either (1) a target site probe to form a transcriptionbubble complex which comprises double-stranded segments on either sideof a single-stranded target site or (2) an abortive promoter cassettecomprising a transcription bubble region which includes a target site or(3) an abortive promoter cassette that is attached to any targetmolecule and then used to generate a signal.

In accordance with one aspect, the invention provides methods ofsynthesizing multiple abortive oligonucleotide transcripts from portionsof a target DNA or RNA sequence, wherein the methods comprise combiningand reacting the following: (a) a single-stranded target nucleic acidcomprising at least one target site; (b) an RNA initiator that iscomplementary to a site on the target nucleic acid that is upstream ofthe target site; (c) an RNA polymerase; (d) optionally, nucleotidesand/or nucleotide analogs; (e) a chain terminator; and (f) optionally,either (1) a target site probe that partially hybridizes to a targetregion on the target nucleic acid, forming a transcription bubblecomplex that includes first and second double-stranded regions on eitherside of a single-stranded target site or (2) an abortive promotercassette comprising a transcription bubble region that includes atranscription start site. The combination is subjected to suitableconditions, as described below, such that (a) a target site probehybridizes with a target nucleic acid in a target region that includesthe target site; (b) an RNA initiator hybridizes upstream of a targetsite; (c) an RNA polymerase utilizes the RNA initiator to initiatetranscription at the target site, elongation occurs, and anoligonucleotide transcript is synthesized; (d) a chain terminatorterminates transcription during elongation; (e) the RNA polymerasereleases the short, abortive oligonucleotide transcript withoutsubstantially translocating from the polymerase binding site ordissociating from the template; and (f) steps (c) through (e) arerepeated until sufficient signal is generated and the reaction isstopped. Alternatively, (a) an abortive promoter cassette hybridizeswith an end of the target nucleic acid; (b) an RNA initiator hybridizesupstream of a transcription start site; (c) an RNA polymerase utilizesthe RNA initiator to initiate transcription at the target site,elongation occurs, and an oligonucleotide transcript is synthesized; (d)a chain terminator terminates transcription during elongation; (e) theRNA polymerase releases the short, abortive oligonucleotide transcriptwithout substantially translocating from the polymerase binding site ordissociating from the template; and (f) steps (c) through (e) arerepeated until sufficient signal is generated and the reaction isstopped.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular BiologyTechniques Manual,” third edition, (Coyne et al., 2001); “ShortProtocols in Molecular Biology,” fourth edition, (Ausubel et al., 1999)“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook etal., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “AnimalCell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology”(Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M.Ausubel et al., eds., 1987, and periodic updates); “PCR: The PolymeraseChain Reaction” (Mullis et al., eds., 1994).

Primers, initiators, oligonucleotides, and polynucleotides employed asreactants in the present invention can be generated using standardtechniques known in the art or may be obtained from commercial sources,including but not restricted to Sigma/Aldrich, Molecular Probes, TrilinkTechnologies.

Terms

To facilitate understanding of the invention, the following terms havethe following meanings unless expressly stated otherwise:

“About” as used herein means that a number referred to as “about”comprises the recited number plus or minus 1-10% of that recited number.For example, “about” 50 nucleotides can mean 45-55 nucleotides or as fewas 49-51 nucleotides depending on the situation.

“Transcription” is an enzyme-mediated process that synthesizes acomplementary RNA transcript that corresponds to a nucleic acid templatesequence. Transcription typically includes three phases, namely,initiation, elongation, and termination. The transcript of the templateis processively synthesized by a polymerase through the formation of aphosphodiester bond between an initiator, which may be a mononucleoside,a mononucleotide, an oligonucleotide, or polynucleotide, and asubsequent NTP, et cetera., without the dissociation of either thenascent transcript or the polymerase from the template, until thepolymerase reaches either a termination sequence on the template or theend of the template sequence or is stopped by other means, such asprotein-DNA transcription roadblocks. As used in typical hybridizationassays, the termination of transcription is generally achieved when thepolymerase completes the elongation phase and reaches the end of thetemplate sequence or a specific transcription termination signal aftertranslocating from the initial enzyme binding site (promoter) on thetemplate. In this context, “translocation” means that the polymerasemoves along the template sequence from an initial enzyme binding site onthe template to another point on the template which is at least 50nucleotides downstream of the enzyme binding site.

“Abortive transcription” is an enzyme-mediated process thatreiteratively initiates and terminates the synthesis of oligonucleotidesthat correspond to at least one portion, or target site, of acomplementary nucleic acid template sequence. The abortiveoligonucleotides synthesized vary in length of nucleotides, and maycontain from about 2 to about 26 nucleotides, about 26 to about 50nucleotides and about 50 nucleotides to about 100 nucleotides, andgreater than 100 nucleotides.

“Abortive transcription” also includes three phases, namely, initiation,elongation, and termination. During the initiation phase, a polymeraseforms a phosphodiester bond between an initiator and a second NTP, andthen adds subsequent NTPs, et cetera., transcribing the templatesequence to synthesize an oligonucleotide transcript of from about 2 toabout 50 nucleotides in length and then terminating the transcriptionevent by releasing the nascent oligonucleotide transcript, without thepolymerase substantially translocating from the polymerase binding siteor dissociating from the template. In other words, the RNA polymerasesubstantially remains at the initial binding site on the template,releases a first nascent oligonucleotide transcript, and then is capableof engaging in another transcription initiation event to produce asecond oligonucleotide transcript, which is substantially complementaryto substantially the same target site that was transcribed to producethe first oligonucleotide transcript. In this manner, the polymerasereiteratively transcribes a single portion of the template (i.e., atarget region) and releases multiple copies of substantially identicalnascent oligonucleotide transcripts.

“Reverse transcription” refers to the transcription of an RNA templateto synthesize complementary DNA (cDNA).

“Reiterative” refers to multiple identical or highly similar copies of asequence of interest.

“Replication” is an enzyme-mediated process which synthesizes acomplementary nucleic acid molecule from a single-stranded nucleic acidtemplate sequence. The DNA replicate of the template is synthesized by aDNA polymerase through the formation of a phosphodiester bond between aprimer and a first deoxyribonucleoside triphosphate (dNTP), followed bythe formation of a second phosphodiester bond between the first dNTP anda subsequent dNTP, et cetera., without the dissociation of either theDNA replicate or the DNA polymerase from the template, until the DNApolymerase reaches either a termination sequence on the template or theend of the template sequence. In a typical DNA primer extensionreaction, replication of the template terminates when the DNA polymerasesynthesizes the entire template sequence after translocating from theinitial enzyme binding site on the template. In this context,“translocation” means that the DNA polymerase moves along the templatesequence from an initial enzyme binding site on the template to anotherpoint on the template which is downstream of the enzyme binding site.

“Oligonucleotide product” refers to the oligonucleotide that issynthesized by the reiterative synthesis reaction of the presentinvention. An oligonucleotide product may be an “oligonucleotidetranscript,” if the polymerization reaction is a transcription reactioncatalyzed by an RNA polymerase, or an “oligonucleotide repeat,” if thepolymerization reaction is a DNA synthesis reaction catalyzed bytelomerase or DNA polymerase.

“Termination” refers to the use of a chain terminator to conclude achain elongation or primer extension reaction that is catalyzed by apolymerase. A “chain terminator” or “terminator” may comprise anycompound, composition, complex, reactant, reaction condition, or processstep (including withholding a compound, reactant, or reaction condition)which inhibits the continuation of transcription by the polymerasebeyond the initiation and/or elongation phases. A “chain terminatingnucleotide” is a chain terminator that comprises a nucleotide ornucleotide analog that inhibits further chain elongation onceincorporated, due to either the structure of the nucleotide analog orthe sequence of the nucleic acid being copied or transcribed.

A “target sequence” or “target polynucleotide” is a polynucleotidesequence of interest for which detection, characterization orquantification is desired. The actual nucleotide sequence of the targetsequence may be known or not known.

A “target site” is that portion of the target sequence that is detectedby transcription by a polymerase to form an oligonucleotide product. Inaccordance with the invention, there is at least one target site on atarget nucleic acid. The sequence of a target site may or may not beknown with particularity. That is, while the actual genetic sequence ofthe target nucleic acid may be known, the genetic sequence of aparticular target site that is transcribed or replicated by a polymeraseneed not be known.

A “target region” is that portion of a target sequence to which a targetsite probe partially hybridizes to form a bubble complex, as describedin detail below. In accordance with the invention, there is at least onetarget region on a target nucleic acid, and each target region comprisesa target site. The sequence of a target region is known with sufficientparticularity to permit sufficiently stringent hybridization of acomplementary target site probe, such that the target site probe forms abubble complex with the target region.

Generally, a “template” is a polynucleotide that contains the targetnucleotide sequence. In some instances, the terms “target sequence”,“template polynucleotide”, “target nucleic acid”, “targetpolynucleotide”, “nucleic acid template”, “template sequence”, andvariations thereof, are used interchangeably. Specifically, the term“template” refers to a strand of nucleic acid on which a complementarycopy is synthesized from nucleotides or nucleotide analogs through theactivity of a template-dependent nucleic acid polymerase. Within aduplex, the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand. The “template” strand mayalso be referred to as the “sense” strand, and the non-template strandas the “antisense” strand.

“Synthesis” generally refers to the process of producing at least onecomplementary copy of a target site or other portion of a targetsequence. “Multiple copies” means at least 2 copies. A “copy” does notnecessarily mean perfect sequence complementarity or identity with thetemplate sequence. For example, copies can include nucleotide analogs,intentional sequence alterations (such as sequence alterationsintroduced through a primer comprising a sequence that is hybridizable,but not complementary, to the template), and/or sequence errors thatoccur during synthesis. “Synthesis” encompasses both transcription of atarget nucleic acid and replication of a target nucleic acid.

“Polynucleotide” or “nucleic acid strand”, as used interchangeablyherein, refers to nucleotide polymers of any length, such as two ormore, and includes both DNA and RNA. The nucleotides can bedeoxyribonucleotides, ribonucleotides, nucleotide analogs (includingmodified phosphate moieties, bases, or sugars), or any substrate thatcan be incorporated into a polymer by a suitable enzyme, such as a DNApolymerase or an RNA polymerase. Thus, a polynucleotide may comprisemodified nucleotides, such as methylated nucleotides, and their analogs.If present, modification to the nucleotide structure may be impartedbefore or after synthesis of the polymer. The sequence of nucleotidesmay be interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications include, for example,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, cabamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,glutathione-s-transferase, methylases, demethylases, DNA repair enzymes,nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.),those with intercalators (e.g., ethidium, acridine, psoralen, etc.),those with antibody-specific haptens (dinitrophenyl (DNP), biotin,etc.), those with affinity tags (hexahistadine, glutathione, etc.),those containing chelators (e.g., metals, radioactive metals, boron,oxidative metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.), those withchemical or photochemical activities (DNA or RNA cleavage agents,crosslinkers, fluorescent compounds, etc.) as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent on the pentose (i.e., ribose or deoxyribose) ring of anucleotide may be, for example, replaced by phosphonate or phosphategroups, protected by standard protecting groups, activated to prepareadditional linkages to additional nucleotides, or conjugated to a solidsupport. The 5′ and 3′ terminal OH groups on the pentose ring of anucleotide can be phosphorylated or substituted with amines or organiccapping group moieties of from about 1 to about 50 carbon atoms. Otherhydroxyl groups on the ribose or deoxyribose ring may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example,2′-O-methyl-2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugaranalogs, anomeric sugars, epimeric sugars, such as arabinose, xylose,pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, andabasic nucleoside analogs such as methyl riboside. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

“Nucleotide” or “NTP” refers to a base-sugar-phosphate compound. “Base”refers to a nitrogen-containing ring molecule that, when combined with apentose sugar and a phosphate group, form a nucleotide. Bases includesingle ring pyrimidines, such as cytosine (C), thymine (T), and uracil(U), and double ring purines, such as adenine (A) and guanine (G).“Sugar” or “pentose sugar” generally refers to a pentose ring, such as aribose ring or deoxyribose ring. Nucleotides are the monomeric subunitsof both types of nucleic acid polymers, that is, RNA and DNA.“Nucleotide” or “NTP” refers to any nucleoside 5′ phosphate, that is,ribonucleoside 5′ phosphates (i.e., mono-, di-, and triphosphates) anddeoxyribonucleoside 5′ phosphates (i.e., mono-, di-, and triphosphates),and includes “nucleoside phosphate analogs”, “nucleotide analogs”, and“NTP analogs”. “Nucleoside phosphate analog”, “nucleotide analog”, and“NTP analog” refer to any nucleoside 5′ phosphate (i.e., mono-, di-, ortriphosphate) which is analogous to a native nucleotide but whichcontains one or more chemical modifications when compared to thecorresponding native nucleotide. Nucleotide analogs includebase-modified analogs (e.g.5-mercapto pyrimidines, 8-mercapto purines),phosphate-modified analogs (e.g., α-thio-triphosphates), andsugar-modified analogs (3′ OMe, 3′deoxy) and may comprise modified formsof deoxyribonucleotides as well as ribonucleotides.

“Nucleoside” refers to a base-sugar combination without a phosphategroup. Nucleosides include, but are note limited to, adenosine (A),cytidine (C), guanosine (G), thymidine (T), and uridine (U).

The term “oligonucleotide” generally refers to short, typicallysingle-stranded, synthetic polynucleotides that are generally, but notnecessarily, less than about 200 nucleotides in length. Moreparticularly, an oligonucleotide may be defined as a molecule comprisedof two or more nucleotides, including deoxyribonucleotides and/orribonucleotides. The exact size depends on many factors, which in turndepend on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, degradation of longer DNA or RNA,transcription, reverse transcription, abortive transcription orreiterative synthesis, as further described herein, and a combinationthereof.

Because mononucleotides undergo a reaction which synthesizesoligonucleotides by covalently bonding the 3′ oxygen of a firstmononucleotide pentose ring to the 5′ phosphate of a secondmononucleotide through a phosphodiester linkage, a first end of anoligonucleotide is referred to as the “5′ end” if the 5′ phosphate ofthe terminal nucleotide is not linked to a 3′ oxygen of a nucleotidepentose ring, and a second end of an oligonucleotide is referred to asthe “3′ end” if the 3′ oxygen of the terminal nucleotide is not linkedto a 5′ phosphate of a subsequent nucleotide pentose ring. As usedherein, a nucleic acid sequence, even if the sequence is internal to alarger oligonucleotide, also may be said to have 5′ and 3′ ends. Forsingle-stranded DNA or RNA, a first region along a nucleic acid strandis said to be “upstream” of a second region, if the 3′ end of the firstregion is before the 5′ end of the second region when moving along astrand of nucleic acid in a 5′→3′ direction. Conversely, a first regionalong a nucleic acid strand is said to be “downstream” of a secondregion, if the 5′ end of the first region is after the 3′ end of thesecond region when moving along a strand of nucleic acid in a 5′→3′direction.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide that is 3′ (downstream) from anotherregion or position in the same polynucleotide or oligonucleotide whenmoving along the polynucleotide or oligonucleotide in a 5′→3′ direction.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide that is 5′ (upstream) from anotherregion or position in the same polynucleotide or oligonucleotide whenmoving along the polynucleotide or oligonucleotide in a 5′→3′ direction.

“Nucleic acid sequence” refers to an oligonucleotide or polynucleotide,and fragments, segments, or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represents either the sense or the antisense strand.

The term “substantially single-stranded”, when used in reference to anucleic acid substrate, means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two substantiallycomplementary segments or regions of nucleic acid that are held togetherby inter-strand or intra-strand base pairing interactions.

As used herein, the terms “complementary” or “complementarity” are usedin reference to a first polynucleotide (which may be an oligonucleotide)which is in “antiparallel association” with a second polynucleotide(which also may be an oligonucleotide). As used herein, the term“antiparallel association” refers to the alignment of twopolynucleotides such that individual nucleotides or bases of the twoassociated polynucleotides are paired substantially in accordance withWatson-Crick base-pairing rules. For example, the sequence “A-G-T” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the polynucleotides' bases are matched accordingto the base pairing rules. Or, there may be “complete” or “total”complementarity between the polynucleotides. The degree ofcomplementarity between the polynucleotides has significant effects onthe efficiency and strength of the hybridization between twopolynucleotides. This is of particular importance in synthesisreactions, as well as detection methods which depend upon bindingbetween polynucleotides. Those skilled in the art of nucleic acidtechnology can determine duplex stability empirically by considering anumber of variables, including, for example, the length of the firstpolynucleotide, which may be an oligonucleotide, the base compositionand sequence of the first polynucleotide, and the ionic strength andincidence of mismatched base pairs. A general formula that may be usedto calcuate the melting temperature of an oligonucleotide is:Tm=(2(UA)+4(GC))−0.5C for every 1% formamide. For DNA-DNA hybrids, theTm is approximated by the following formula: Tm=81.5+16.6 (log M)+0.41(% G+C)−500/L; M is the molarity of the monovalent cations; L is thelength of the hybrid base pairs (Anal Biochem. 138:267-284, 1984).

The terms “self-complementary” and “self-complementarity”, when used inreference to a polynucleotide (e.g., an oligonucleotide), mean thatseparate regions of the polynucleotide can base-pair with each other.Because this term refers only to intramolecular base-pairing, any strandsaid to have a region of self-complementarity must have at least tworegions capable of base-pairing with one another. As defined above,complementarity may be either “complete” or “partial”. As used inreference to the oligonucleotides of the present invention, regions ofan oligonucleotide are considered to have significantself-complementarity when these regions are capable of forming a duplexof at least 3 contiguous base pairs (i.e., three base pairs of completecomplementarity), or when they may form a longer duplex that ispartially complementary.

The term “primer” generally refers to a short, single-strandedoligonucleotide which has a free 3′-OH group and which can bind to andhybridize with a target sequence that is potentially present in a sampleof interest. After hybridizing to a target sequence, a primer is capableof promoting or initiating polymerization or synthesis of apolynucleotide or oligonucleotide extension product that iscomplementary to the target sequence or a portion of the targetsequence. A primer is selected to be “substantially” complementary to aspecific portion of a target nucleic acid sequence. A primer issufficiently complementary to hybridize with a target sequence andfacilitate either transcription or replication of a portion of thetarget nucleic acid. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the templatestrand. Non-complementary bases can be interspersed within the primer,provided that the primer sequence has sufficient complementarity withthe template sequence to hybridize with the template and thereby form atemplate-primer complex for initiating synthesis of a polynucleotide oroligonucleotide product.

The term “initiator” refers to a mononucleoside, mononucleotide,oligonucleotide, polynucleotide or analog thereof, which is incorporatedinto the 5′ end of a nascent RNA molecule and may be considered a“primer” for RNA synthesis (“initiator primer”).

In one embodiment, an RNA initiator facilitates the initiation oftranscription at a target site on a single-stranded target nucleic acidin the absence of a template promoter sequence, as is known in the art.(See, U.S. Pat. No. 5,571,669; Daube and von Hippel, Science, 258:1320-1324 (1992)). In another embodiment, initiators are used torandomly start abortive transcription at a plurality of target sites onthe nucleic acid template (FIG. 16). The initiators and/or theindividual nucleotides or nucleotide analogs that are used to extend theinitiators may be suitably modified to enable signal generation,detection of the oligonucleotide products, and a determination of thepresence or absence of the target sequence.

For example, it may be desirable to modify the initiator to provide theinitiator with a label moiety for a variety of purposes, includingdetection of the abortive oligonucleotide product(s). Examples of suchmodifications include, but are not limited to, fluorescent molecules andenergy transfer dyes (such as, fluorescein, aedans, coumarine, bodipydyes, and rhodamine based dyes), fluorescent quencher molecules (forexample, Dabcyl), proteins, peptides, amino linkers, or amino acid basedmolecules (for example polyhistidine), modified bases and modified andunmodified base analogs, peptide nucleic acids (PNAs),methylphosphonates, radioactive labels, terminal phosphates, 3′glyceryl, other carbohydrate based molecules, fatty acid derivedmolecules, carbon spacer molecules, electrochemiluminescent labels,lanthamide labels, avidin and its derivatives (for example,streptavidin, Neutravidin, etc.), biotin, steroid molecules (such asDigoxygenin), thiol linkages, ferritin labels, and the like.

As used herein, the term “hybridization” is used in reference to thebase-pairing of complementary nucleic acids, including polynucleotidesand oligonucleotides. Hybridization and the strength of hybridization(i.e., the strength of the association between the nucleic acids) isimpacted by such factors as the degree of complementary between thenucleic acids, the stringency of the reaction conditions involved, themelting temperature (T_(m)) of the formed hybrid, and the G:C ratiowithin the duplex nucleic acid. Generally, “hybridization” methodsinvolve annealing a complementary polynucleotide to a target nucleicacid (i.e., the sequence to be detected either by direct or indirectmeans). The ability of two polynucleotides and/or oligonucleotidescontaining complementary sequences to locate each other and anneal toone another through base pairing interactions is a well-recognizedphenomenon.

With regard to complementarity, it may be important for some diagnosticapplications to determine whether the hybridization of twopolynucleotides and/or oligonucleotides represents complete or partialcomplementarity. For example, where it is desired to detect simply thepresence or absence of pathogen DNA (such as from a virus, bacterium,fungi, mycoplasma, or protozoan for example), the hybridization methodneed only ensure that hybridization occurs when the relevant sequence ispresent; conditions can be selected where both partially complementaryprobes and completely complementary probes will hybridize. Otherdiagnostic applications, however, may require that the hybridizationmethod be capable of distinguishing between partial and completecomplementarity, such as in cases where it may be of interest to detecta genetic polymorphism, that is, a difference in a single base pairbetween multiple alleles (variations) that may exist for a particulargene or genetic marker.

“Stringency” generally refers to the conditions under which nucleic acidhybridizations are conducted, including temperature, ionic strength, andthe presence of other compounds. Conditions of “high stringency”generally refer to those conditions under which nucleic acid basepairing will occur only between polynucleotide and/or oligonucleotideregions that have a high frequency of complementary base sequences.Consequently, conditions of “weak” or “low” stringency may be preferredwhen it is desirable to hybridize or anneal two polynucleotides and/oroligonucleotides, which are not completely complementary to one another.

The term “reactant” is used in its broadest sense. A reactant cancomprise an enzymatic reactant, a chemical reactant, or ultravioletlight (ultraviolet light, particularly short wavelength ultravioletlight, is known to break polynucleotide polymers). Any agent capable ofreacting with an oligonucleotide or polynucleotide to modify theoligonucleotide or polynucleotide is encompassed by the term “reactant,”including a “reactant nucleotide” that is added to a reaction mixturefor incorporation into an oligonucleotide product by a polymerase.

A “complex” is an assembly of components. A complex may or may not bestable and may be directly or indirectly detected. For example, asdescribed herein, given certain components of a reaction and the type ofproduct(s) of the reaction, the existence of a complex can be inferred.For the purposes of this invention, a complex is generally anintermediate with respect to a final reiterative synthesis product, suchas a final abortive transcription or replication product for example.

A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

The term “enzyme binding site” refers to a polynucleotide region that ischaracterized by a sequence or structure that is capable of binding to aparticular enzyme or class of enzymes, such as a polymerase.

“Polymerase” refers to any agent capable of facilitating or catalyzingthe polymerization (joining) of nucleotides and/or nucleotide analogs.Suitable agents include naturally occurring enzymes, such as naturallyoccurring RNA polymerases (including RNA-dependent and DNA-dependent RNApolymerases), DNA polymerases (including DNA-dependent and RNA-dependentDNA polymerases), as well as modified or mutant enzymes that maycurrently exist (such as the mutant RNA polymerases disclosed in Sousa,et al., U.S. Pat. No. 6,107,037 for example) or may be hereafter createdor designed, which modified or mutant enzymes may be designed to exhibitcharacteristics that are desirable for particular applications.Exemplary characteristics of a modified or mutant enzyme may include,but are not limited to, relaxed template specificity, relaxed substratespecificity, increased thermostability, and/or the like. It is intendedthat the term “polymerase” encompasses both thermostable andthermolabile enzymes.

The term “thermostable” when used in reference to an enzyme, such as anRNA or DNA polymerase for example, indicates that the enzyme isfunctional or active (i.e., can perform catalysis) at an elevatedtemperature, that is, at about 55° C. or higher. Thus, a thermostablepolymerase can perform catalysis over a broad range of temperatures,including temperatures both above and below about 55° C.

The term “template-dependent polymerase” refers to a nucleic acidpolymerase that synthesizes a polynucleotide or oligonucleotide productby copying or transcribing a template nucleic acid, as described above,and which does not synthesize a polynucleotide in the absence of atemplate. This is in contrast to the activity of a template-independentnucleic acid polymerase, such as terminal deoxynucleotidyl transferaseor poly-A polymerase for example, that may synthesize or extend nucleicacids in the absence of a template.

A “DNA-dependent RNA polymerase” is an enzyme which facilitates orcatalyzes the polymerization of RNA from a complementary DNA template.

A “DNA-dependent DNA polymerase” is an enzyme which facilitates orcatalyzes DNA replication or synthesis, that is, the polymerization ofDNA from a complementary DNA template.

An “RNA-dependent RNA polymerase” is an enzyme which facilitates orcatalyzes the polymerization of RNA from a complementary RNA template.

An “RNA-dependent DNA polymerase” or “reverse transcriptase” is anenzyme that facilitates or catalyzes the polymerization of DNA from acomplementary RNA template.

“Primer extension”, “extension”, “elongation”, and “extension reaction”is the sequential addition of nucleotides to the 3′ hydroxyl end of amononucleotide, oligonucleotide, or polynucleotide initiator or primerwhich has been annealed or hybridized to a longer, templatepolynucleotide, wherein the addition is directed by the nucleic acidsequence of the template and/or the binding position of the polymerase.Extension generally is facilitated by an enzyme capable of synthesizinga polynucleotide or oligonucleotide product from a primer or initiator,nucleotides and a template. Suitable enzymes for these purposes include,but are not limited to, any of the polymerases described above.

“Incorporation” refers to becoming a part of a nucleic acid polymer.There is a known flexibility in the terminology regarding incorporationof nucleic acid precursors. For example, the nucleotide dGTP is adeoxyribonucleoside triphosphate. Upon incorporation into DNA, dGTPbecomes dGMP, that is, a deoxyguanosine monophosphate moiety. AlthoughDNA does not include dGTP molecules, one may say that one incorporatesdGTP into DNA.

The terms “sample” and “test sample” are used in their broadest sense.For example, a “sample” or “test sample” is meant to include a specimenor culture (e.g., microbiological cultures) as well as both biologicaland environmental samples. Samples of nucleic acid used in the methodsof the invention may be aqueous solutions of nucleic acid derived from abiological or environmental sample and separated, by methods known inthe art, from other materials, such as proteins, lipids, and the like,that may be present in the sample and that may interfere with themethods of the invention or significantly increase the “background”signal in carrying out the methods.

A biological sample may comprise any substance which may include nucleicacid, such as animal (including human) tissue, animal fluids (such asblood, saliva, mucusal secretions, semen, urine, sera, cerebral orspinal fluid, pleural fluid, lymph, sputum, fluid from breast lavage,and the like), animal solids (e.g., stool), cultures of microorganisms,liquid and solid food and feedproducts, waste, cosmetics, or water thatmay be contaminated with a microorganism, or the like. An environmentalsample may include environmental material, such as surface matter, soil,water, and industrial samples, as well as samples obtained from food anddairy processing instruments, apparatus, equipment, utensils, anddisposable and non-disposable items. These examples are merelyillustrative and are not intended to limit the sample types applicableto the present invention.

“Purified” or “substantially purified” refers to nucleic acids that areremoved from their natural environment, isolated or separated, and areat least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which they are naturally associated. An“isolated polynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence, so long as thedesired functional activity is retained.

A “deletion” is defined as a change in a nucleic acid sequence in whichone or more nucleotides are absent as compared to a standard nucleicacid sequence.

An “insertion” or “addition” is a change in a nucleic acid sequencewhich has resulted in the addition of one or more nucleotides ascompared to a standard nucleic acid sequence.

A “substitution” results from the replacement of one or more nucleotidesin a nucleic acid by different nucleotides.

An “alteration” in a nucleic acid sequence refers to any change in anucleic acid sequence or structure, including, but not limited to adeletion, an addition, an addition-deletion, a substitution, aninsertion, a reversion, a transversion, a point mutation, or amicrosatellite alteration, or methylation.

“Methylation” refers to the addition of a methyl group (—CH₃) to anucleotide base in DNA or RNA.

Sequence “mutation” refers to any sequence alteration in a sequence ofinterest in comparison to a reference sequence. A reference sequence canbe a wild type sequence or a sequence to which one wishes to compare asequence of interest. A sequence mutation includes single nucleotidechanges, or alterations of more than one nucleotide in a sequence, dueto mechanisms such as substitution, deletion, or insertion. A singlenucleotide polymorphism (SNP) is also a sequence mutation as usedherein.

“Microarray” and “array,” as used interchangeably herein, refer to anarrangement of a collection of polynucleotide sequences in a centralizedlocation. Arrays can be on a solid substrate, such as a glass slide, oron a semi-solid substrate, such as nitrocellulose membrane. Thepolynucleotide sequences can be DNA, RNA, or any combinations thereof.

The term “label” refers to any atom, molecule, or moiety which can beused to provide a detectable (preferably quantifiable) signal, eitherdirectly or indirectly, and which can be attached to a nucleotide,nucleotide analog, nucleoside mono-, di-, or triphosphate, nucleosidemono-, di-, or triphosphate analog, polynucleotide, or oligonucleotide.Labels may provide signals that are detectable by fluorescence,radioactivity, chemiluminescence, electrical, paramagnetism,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like. A label may be a charged moiety(positive or negative charge) or, alternatively, may be charge neutral.

“Detection” includes any means of detecting, including direct andindirect detection. For example, “detectably fewer” products may beobserved directly or indirectly, and the term indicates any reduction inthe number of products (including no products). Similarly, “detectablymore” products means any increase, whether observed directly orindirectly.

As used herein, the terms “comprises,” “comprising”, “includes”, and“including”, or any other variations thereof, are intended to cover anonexclusive inclusion, such that a process, method, composition,reaction mixture, kit, or apparatus that comprises a list of elementsdoes not include only those elements but may include other elements notexpressly listed or inherent to such process, method, composition,reaction mixture, kit, or apparatus.

“A,” “an,” “the,” and the like, unless otherwise indicated, includeplural forms.

Components and Reaction Conditions

Target Nucleic Acid

The target nucleic acid can be either a naturally occurring or syntheticpolynucleotide segment, and it can be obtained or synthesized bytechniques that are well-known in the art. A target sequence to bedetected in a test sample may be present initially as a discretemolecule, so that the sequence to be detected constitutes the entirenucleic acid, or may be present as only one component of a largermolecule. The target nucleic acid can be only a minor fraction of acomplex mixture, such as a biological sample, and can be obtained fromvarious biological materials by procedures that are well-known in theart. The target nucleic acid to be detected may include nucleic acidsfrom any source, in purified, or unpurified form, which can be DNA(including double-stranded (ds) DNA and single-stranded (ss) DNA) or RNA(including tRNA, mRNA, rRNA), mitochondrial DNA or RNA, chloroplast DNAor RNA, DNA-RNA hybrids, or mixtures thereof; genes, chromosomes, orplasmids; and the genomes of biological material, such as the genomes ofmicroorganisms (including bacteria, yeast, viruses, viroids, molds, andfingi), plants, animals, humans, or fragments thereof. Standardtechniques in the art are used to obtain and purify the nucleic acidsfrom a test sample. Methods for the extraction and/or purification ofsuch nucleic acids have been described, for example, by Sambrook, etal., Molecular Cloning: A Laboratory Manual (New York, Cold SpringHarbor Laboratory, third edition, 2000). Detection of an RNA target mayor may not require initial complementary DNA (cDNA) synthesis, as knownin the art. Detection of a DNA-RNA hybrid may require denaturation ofthe hybrid to obtain a ssDNA or denaturation followed by reversetranscription to obtain a cDNA.

Target Proteins

In another embodiment of the invention, the target may be anothermolecule, such as a protein, which is labeled by covalent or noncovalentattachment of a defined nucleic acid sequence which can be used forreiterative oligonucleotide synthesis (FIG. 23). The target protein canbe either a naturally occurring or synthetic polypeptide segment, and itcan be obtained or synthesized by techniques that are well-known in theart. A target protein to be detected in a test sample may be presentinitially as a discrete molecule, so that the protein to be detectedconstitutes the entire protein, or may be present as only one componentof a larger complex. The target protein can be only a minor fraction ofa complex mixture, such as a biological sample, and can be obtained fromvarious biological materials by procedures that are well-known in theart. The target protein to be detected may include proteins from anysource, in purified or unpurified form. Standard techniques in the artare used to obtain and purify the proteins from a test sample. Methodsfor the extraction and/or purification of such proteins have beendescribed, for example, by Sambrook, et al., Molecular Cloning: ALaboratory Manual (New York, Cold Spring Harbor Laboratory, thirdedition, 2000).

Immobilization

In one embodiment of the invention, the target molecule may beimmobilized. In another embodiment, the target molecule may beimmobilized to form, for example, a microarray. A single molecule arrayin accordance with this embodiment includes a solid matrix, abioreactive or bioadhesive layer, and a bioresistant layer. Solid phasesthat are useful as a matrix for the present invention include, but arenot limited to, polystyrene, polyethylene, polypropylene, polycarbonate,or any solid plastic material in the shape of test tubes, beadsmicroparticles, dip-sticks, or the like. Additionally, matrices include,but are not limited to, membranes, microtiter plates (e.g., 96-well and384-well), test tubes, and Eppendorf tubes. Solid phases also includeglass beads, glass test tubes, and any other appropriate shape that ismade of glass. A functionalized solid phase, such as plastic or glass,which has been modified so that the surface carries carboxyl, amino,hydrazide, or aldehyde groups can also be used. In general, suitablesolid matrices comprise any surface to which a bioadhesive layer, suchas a ligand-binding agent, can be attached or any surface which itselfprovides a ligand attachment site.

The bioadhesive layer can be an ionic adsorbent material such as gold,nickel, or copper (Montemagno and Bachand, Constructing NanomechanicalDevices Powered by Biomolecular Motors, Nanotechnology, 10: 225-231(1999)), protein-adsorbing plastics, such as polystyrene (U.S. Pat. No.5,858,801), or a covalent reactant, such as a thiol group. To create apatterned array in the bioadhesive layer, an electron-sensitive polymer,such as polymethyl methacrylate (PMMA) for example, can be used to coatthe solid support and can be etched in any desired pattern with anelectron beam followed by development to remove the sensitized polymer.The etched portions of the polymer are then coated with a metal, such asnickel, and the polymer is removed with a solvent, leaving a pattern ofmetal posts on the substrate. This method of electron beam lithographyprovides the high spatial resolution and small feature size whichfacilitates the immobilization of a single molecule at each point in thepatterned array. An alternate means for creating high-resolutionpatterned arrays is atomic force microscopy. A further means is X-raylithography.

Antibody or oligonucleotide capture probes can be attached to thebioadhesive pattern by providing a polyhistidine tag on the captureprobe that binds to the metal bioadhesive patterns. The capture probesmay be, for example, from about 15 to about 500 nucleotides in length.Other conventional means for attachment employ homobifunctional andheterobifunctional crosslinking reagents. Homobifunctional reagentscarry two identical functional groups, whereas heterobifunctionalreagents contain two dissimilar functional groups to link the captureprobes to the bioadhesive. The heterobifunctional cross-linking agentsmay contain a primary amine-reactive group and a thiol-reactive group.Covalent crosslinking agents are selected from reagents capable offorming disulfide (S—S), glycol (—CH(OH)—CH(OH)—), azo (—N═N—), sulfone(—S(═O₂—), ester (—C(═O)—O—), or amide (—C(═O)—N—) bridges. Crosslinkingagents include, but are not limited to, maleamides, iodoacetamides, anddisulfies. Table 1 provides a list of representative classes ofcrosslinking reagents and their group specificity (Wong, S. S. Chemistryof Protein Conjugation and Cross-Linking, 1991, CRC Press, Inc., BocaRaton, USA).

TABLE 1 Crosslinking Reagents and group specificity Reagent Groupspecificity alpha-haloacetyl compounds eg SH, S—CH₃, NH₂, phenolic,ICH2COOH imadazole N-maleimides SH, NH₂ mercurials SH Disulfides SH Arylhalides SH, NH₂, phenolic, imidazole Acid anhydrides eg. Succinicanhydride NH₂, phenolic Isocyanates eg. HNCO NH₂ Isothiocyanates R-NCSNH₂ Sulfonyl halides NH₂ Imidoesters NH₂ Diazoacetates COOH, SHDiazonium salts eg benzene-N2+ Cl- phenolic, imidazole dicarbonylcompound NH—C(NH)—NH₂

A bioresistant layer may be placed or superimposed upon the bioadhesivelayer either before or after attachment of the capture probe to thebioadhesive layer. The bioresistant layer is any material that does notbind the capture probe. Non-limiting examples include bovine serumalbumin, gelatin, lysozyme, octoxynol, polysorbate 20(polyethenesorbitan monolaurate), and polyethylene oxide containingblock copolymers and surfactants (U.S. Pat. No. 5,858,801). Depositionof the bioadhesive and bioresistant layers may be accomplished byconventional means, including spraying, immersion, and evaporativedeposition (metals).

In one embodiment, the solid matrix may be housed in a flow chamberhaving an inlet and outlet to accommodate the multiple solutions andreactants that are allowed to flow past the immobilized capture probes.The flow chamber can be made of plastic or glass and may be either openor transparent in the plane viewed by a microscope or optical reader.Electro-osmotic flow includes a fixed charge on the solid support and avoltage gradient (current) passing between two electrodes placed atopposing ends of the solid support.

Primers

In accordance with the invention, a primer is used to initiatereplication by a DNA polymerase of a target site on the target nucleicacid. If the polymerase is a DNA polymerase, the primer may be comprisedof ribonucleotides or deoxyribonucleotides. The primers and/or theindividual nucleotides or nucleotide analogs that are used to extend theprimers may be suitably modified to enable signal generation, detectionof the oligonucleotide products, and a determination of the presence orabsence of the target sequence.

The primers used in the practice of the invention may be madesynthetically, using conventional chemical or enzymatic nucleic acidsynthesis technology. In one embodiment, the primers are less than about25 nucleotides in length, usually from about 1 to about 10 nucleotidesin length, and preferably about 2 to 3 nucleotides in length. It may bedesirable to modify the nucleotides or phosphodiester linkages in one ormore positions of the primer. Examples of such modifications include,but are not limited to, fluorescent molecules and energy transfer dyes(such as, fluorescein, aedans, coumarine, bodipy dyes, and rhodaminebased dyes), fluorescent quencher molecules (for example, Dabcyl),proteins, peptides, amino linkers, or amino acid based molecules (forexample polyhistidine), modified bases and modified and unmodified baseanalogs, peptide nucleic acids (PNAs), methylphosphonates, radioactivelabels, terminal phosphates, 3′ glyceryl, other carbohydrate basedmolecules, fatty acid derived molecules, carbon spacer molecules,electrochemiluminescent labels, lanthamide labels, avidin and itsderivatives (for example, streptavidin, Neutravidin, etc.), biotin,steroid molecules (such as Digoxygenin), thiol linkages, ferritinlabels, and the like.

Target Site Probes

In accordance with the invention, an oligonucleotide target site probeis used to direct a polymerase to a target site on the target nucleicacid by forming a bubble complex in a target region of the targetnucleic acid (FIG. 11). The target site probe may vary in the length ofnucleotides, including but not limited to, about 20 to about 50nucleotides, about 51 to about 75 nucleotides, about 76 to about 100nucleotides, and greater than 100 nucleotides. The bubble complexcomprises double-stranded regions on either side of a single-strandedregion which includes a target site. In one embodiment, the target siteprobe includes three regions: a first region on the 5′ end of the targetsite probe is complementary to and hybridizes with the template sequenceupstream of a target site-on the template sequence; a second region,which is 3′ of the first region, is non-complementary to the templatesequence and therefore does not hybridize with the template sequence;and a third region, which is on the 3′ end of the target site probe, iscomplementary to and hybridizes with the template sequence downstream ofthe target site. The target site probe can vary in nucleotide length,including but not limited to, about 5-19; about 20 to about 50nucleotides, about 51 to about 75 nucleotides, about 76 to about 100nucleotides and greater than 100 nucleotides.

Use of the target site probe directs the polymerase to a particularenzyme binding site (i.e., the double-stranded segment and bubble formedupstream of the target site by the template sequence and the primer) onthe template sequence to facilitate the initiation of transcription at aparticular target site. That is, rather than facilitating the randominitiation of synthesis reactions by the polymerase along the length ofa single-stranded template sequence, as described above, this embodimentprovides targeted binding of the polymerase for the detection of aparticular target site encompassed by the bubble complex formed by thetarget site probe.

The target site probes used in the practice of the invention may be madeenzymatically or synthetically, using conventional nucleic acidsynthesis technology, such as phosphoramidite, H-phosphonate, orphosphotriester chemistry, for example. Alternative chemistries, such asthose which result in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, may also be employed.The target site probes may be ordered commercially from a variety ofcompanies which specialize in custom polynucleotides and/oroligonucleotides, such Operon, Inc. (Alameda, Calif.).

The sequence of the target site probe will vary depending upon thetarget sequence. The overall length of the target site probe is selectedto provide for hybridization of the first and third regions with thetarget sequence and optimization of the length of the second,non-hybridized region. The first and third regions of the target siteprobe are designed to hybridize to known internal sites on the targetnucleic acid template. Depending upon the application, the sequence ofthe second region on the target site probe can be designed such that thesecond region may or may not be self-complementary. The overall lengthof the target site probe ranges from about 20 to about 50 nucleotides,preferably from about 25 to about 35 nucleotides. The first and thirdregions of the target site probe each range from about 5 to about 20nucleotides in length, preferably from about 8 to about 10 nucleotidesin length. In one embodiment, the first and third regions of the targetsite probe are each about 10 nucleotides in length. The internal, secondregion on the target site probe ranges in length from about 8 to about14 nucleotides, preferably from about 12 to about 14 nucleotides.

In one embodiment, at least one target site probe is used tospecifically initiate abortive oligonucleotide synthesis at one or moretarget sites on the nucleic acid template to produce multipleoligonucleotide products. In another embodiment, the target site probedirects the initiation of abortive transcription on a single-strandedtarget site in the absence of a template promoter sequence, as is knownin the art. (See, U.S. Pat. No. 5,571,669; Daube and von Hippel,Science, 258: 1320-1324 (1992)).

Abortive Promoter Cassette

In accordance with the invention, an abortive promoter cassette (APC)may be used to link a target to a defined sequence to generate multipledetectable oligonucleotide products that indicate the presence of thetarget in a test sample. The APC is a self-complementary sequence of DNAthat may consist of: (1) one contiguous oligonucleotide to which RNApolymerase can bind to form a transcription bubble; (2) two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; or (3) two complementaryoligonucleotides that form a transcription bubble region in the presenceof an RNA polymerase, which allows for the synthesis of an abortiveoligonucleotide product. The APC may contain an artificial promoter, orit may contain the promoter for a specific RNA polymerase. For example,trinucleotide or tetranucleotide products that could be generated fromwith a common phage RNA polymerase can be made with a labeled GpA orGpApA initiator and a labeled pppG or pppA terminator.

In an exemplary embodiment, as illustrated in FIG. 1, the APC compriseseight regions, including an APC linker sequence which comprises either a3′ or a 5′ single-stranded overhang region (i.e., a “sticky end”). Afirst region (A) on the 5′ end of the APC is complementary to a secondregion (A′) near the 3′ end of the APC. A third region (B) and a fourthregion (E) are separated from each other by regions C, D, and C′ and arenon-complementary to each other, such that the regions B and E form asingle-stranded bubble region on the APC when the self-complementaryregions of the APC interact with one another. Regions C and C′ aresubstantially self-complementary, such that the 5′ end of region C iscomplementary to the 3′end of the region C′. Region D may be a shortsequence joining C and C′ for a contiguous APC or may be a regioncomprising the free 3′ or 5′ ends of two separate upper and loweroligonucleotides for a two-part APC. Finally, the APC also includes anAPC linker, a single-stranded region on either the 5′ end or the 3′ endof the APC oligonucleotide, which is formed through the complementaryinteraction of regions A and A′. The APC linker facilitates attachmentof the APC with other target molecules, such as captured target DNA,RNA, or protein, for example.

The APC used in the practice of the invention may be made enzymaticallyor synthetically, using conventional nucleic acid synthesis technology,such as phosphoramidite, H-phosphonate, or phosphotriester chemistry,for example. Alternative chemistries, such as those that result innon-natural backbone groups, such as phosphorothioate, phosphoramidate,and the like, may also be employed. The APC may be ordered commerciallyfrom a variety of companies that specialize in custom polynucleotidesand/or oligonucleotides, such as Operon, Inc. (Alameda, Calif.).

The length of the APC is selected to optimize the stability of thebubble region and provide for the hybridization of the APC linkersequence with the target sequence. The overall length of the APC mayrange from about 50 to about 150 nucleotides, preferably from about 55to about 125 nucleotides. Regions A and A′ may each comprise from about5 to about 25 nucleotides and preferably comprise from about 7 to about15 nucleotides. Regions B and E may comprise from about 8 to about 16nucleotides and preferably comprise from about 10 to about 14nucleotides. Regions C and C′ may each comprise from about 5 to about 25nucleotides and preferably comprise from about 10 to about 20nucleotides. The single-stranded overhang region may comprise from about5 to about 40 nucleotides and preferably comprises from about 10 toabout 25 nucleotides.

Polymerase

Template-dependent polymerases for use in the methods and compositionsof the present invention are known in the art. Either eukaryotic orprokaryotic polymerases may be used. In one embodiment, thetemplate-dependent polymerase is a thermostable polymerase. In anotherembodiment, the polymerase is able to tolerate label moieties on thephosphate group, the nuclease, and/or on the pentose ring ofunincorporated nucleotides. In one embodiment, the polymerase is aDNA-dependent RNA polymerase which is capable of transcribing asingle-stranded DNA template without a promoter sequence. In anotherembodiment, the polymerase is a DNA-dependent RNA polymerase which iscapable of transcribing a single-stranded DNA template having a promotersequence that is capable of binding the particular RNA polymerase beingused. In another embodiment, the polymerase is a DNA-dependent DNApolymerase that is capable of replicating a DNA target site to form aDNA oligonucleotide product. In a further embodiment, the polymerase isan RNA-dependent DNA polymerase that is capable of synthesizing asingle-stranded complementary DNA transcript from an RNA template.Examples of suitable polymerases include the RNA polymerases encoded byEscherichia coli, Escherichia coli bacteriophage T7, Escherichia colibacteriophage T3, and Salmonella typhimurium bacteriophage SP6;RNA-dependent RNA polymerases, such as poliovirus RNA polymerase;reverse transcriptases, such as HIV reverse transcriptase; and DNApolymerases such as Escherichia coli, T7, T4 DNA polymerase, Taqthermostable DNA polymerase, terminal transferase, primase, andtelomerase.

In general, the enzymes included in the methods of the present inventionpreferably do not produce substantial degradation of the nucleic acidcomponents produced by the methods.

Nucleotides

In accordance with the invention, the polymerase catalyzes a reaction inthe usual 5′→3′ direction on the oligonucleotide product and eithertranscribes or replicates the target nucleic acid by extending the 3′end of the initiator or primer through the sequential addition ofnucleotides (NTPs), which may include nucleotide analogs (NTP analogs)and which may be labeled or unlabeled. To facilitate reiterative,abortive synthesis initiation events, the NTPs and/or NTP analogs thatare added to the reaction mixture before and/or during the synthesisreaction include a chain terminator, which is capable of terminating thesynthesis event initiated by the polymerase. Use of the chain terminatorstalls the polymerase during the synthesis reaction, inhibits formationof a processive elongation complex, and thereby promotes the reiterativesynthesis of short abortive oligonucleotides from the target site.(Daube and von Hippel, Science, 258:1320-1324 (1992)).

In accordance with the invention, a chain terminator may comprise anycompound, composition, complex, reactant, reaction condition, or processstep (including withholding a compound, reactant, or reaction condition)which is capable of inhibiting the continuation of transcription orreplication by the polymerase during the primer extension reaction. Inone embodiment, a suitable chain terminator is NTP deprivation, that is,depriving the polymerase of the particular NTP that corresponds to thesubsequent complementary nucleotide of the template sequence. In otherwords, since NTP requirements for chain elongation are governed by thecomplementary strand sequence, given a defined template sequence and adefined primer length, a selected NTP may be withheld from the reactionmixture such that termination of chain elongation by the polymeraseresults when the reaction mixture fails to provide the polymerase withthe NTP that is required to continue transcription or replication of thetemplate sequence.

Alternatively, in another embodiment, the chain terminator may includenucleotide analogs, which may be labeled or unlabeled and which, uponincorporation into an oligonucleotide product by the polymerase, effectthe termination of nucleotide polymerization. Specifically, since chainelongation by a polymerase requires a 3′ OH for the addition of asubsequent nucleotide, nucleotide analogs having a suitably modified 3′end will terminate chain elongation upon incorporation into theoligonucleotide product. Nucleotide analogs having chain terminatingmodifications to the 3′ carbon of the pentose sugar are known in the artand include nucleotide analogs such as 3′ dideoxyribonucleosidetriphosphates (ddNTPs) and 3′ O-methylribonucleoside 5′ triphosphates,as well as nucleotide analogs having either a —H or a —CH₂ moiety on the3′ carbon of the pentose ring. Alternatively, in a further embodiment,the chain terminator may include nucleotide analogs, either labeled orunlabeled, which have a 3′ OH group, but which, upon incorporation intothe oligonucleotide product, still effect chain termination at somepositions, as described herein (Costas, Hanna, et al., Nucleic AcidsResearch 28: 1849-58 (2000); Hanna, M., Meth Enzymology 180: 383-409(1989); Hanna, M., Nucleic Acids Research 21: 2073-79 (1993); Hanna, M.et al., Nucleic Acid Research 27: 1369-76 (1999)).

NTPs and/or NTP analogs that can be employed to synthesize abortiveoligonucleotide products in accordance with the methods of the inventionmay be provided in amounts ranging from about 1 to about 5000 μM,preferably from about 10 to about 2000 μM. In a preferred aspect,nucleotides and/or nucleotide analogs, such as ribonucleosidetriphosphates or analogs thereof, that can be employed to synthesizeoligonucleotide RNA transcripts by the methods of the invention may beprovided in amounts ranging from about 1 to about 6000 μM, preferablyfrom about 10 to about 5000 μM.

Labeling and Detection

In accordance with an aspect of the invention, detectableoligonucleotide products are synthesized from a target nucleic acidtemplate. The detection and identification of the oligonucleotideproducts are facilitated by label moieties on the initiator and/or onthe NTPs or NTP analogs that are incorporated by the polymerase intoeach oligonucleotide product that is synthesized on the target nucleicacid and/or on other molecules which are part of the synthetic complexor which interact with one or more components of the synthetic complex.The label or reporter moieties may be chemically or enzymaticallyincorporated into the nucleotides forming the primer and/or into thereactant NTPs or NTP analogs that are utilized by the polymerase duringthe extension reaction, or other molecules, and may include, forexample, fluorescent tags; paramagnetic groups; chemiluminescent groups;metal binding sites; intercalators; photochemical crosslinkers;antibody-specific haptens; metals; small molecules which are members ofa specific binding pair (such as biotin and streptavidin for example);and any other reporter moiety or moieties which can produce a detectableand/or quantifiable signal either directly or indirectly. Exemplarynucleotide analogs may include, for example, 8-modified purines(8-APAS-ATP) (Costas, Hanna, et al., Nucleic Acids Research 28: 1849-58(2000)); 5-modified pyrimidines (5-APAS-UTP; 5-APAS-CTP) (Hanna, M.,Meth Enzymology 180: 383-409 (1989); Hanna, M., Nucleic Acids Research21: 2073-79 (1993)); fluorescent ribonucleotides (5-SF-UTP) (Hanna, M.et al., Nucleic Acid Research 27: 1369-76 (1999)); and hapten-taggeddeoxynucleotide precursors (5-DNP-SdU) (Meyer and Hanna, BioconjugateChem 7: 401-412 (1996); U.S. Pat. Nos. 6,008,334 and 6,107,039).

In one embodiment, a fluorophore moiety is attached to the 5′ end of theinitiator that is used to initiate transcription of the target nucleicacid. In another embodiment, a fluorophore moiety is attached to the 5or 8 position of the base of an NTP or NTP analog that is used by thepolymerase to extend the initiator primer. In a further embodiment, afirst fluorophore moiety is attached to the initiator and a secondfluorophore is attached to an NTP or NTP analog that is used to extendthe initiator. In this latter embodiment, a fluorescent energy transfermechanism can be used, wherein the first fluorophore (e.g. fluorescein,aedans, coumarin, etc.) is excited and the emission is read from thesecond fluorophore (e.g. fluorescein, aedans, coumarin, etc.) when thesecond fluorophore is brought into proximity with the first fluorophoreby the polymerase during synthesis of the oligonucleotide product.Alternatively, the first and second fluorophores may function by anelectron transfer mechanism, wherein the first fluorophore absorbsenergy from the second fluorophore when the polymerase brings the firstand second fluorophores into proximity with each other, and the firstfluorophore releases the energy in a radiative manner, thereby enablingdetection.

In one aspect, a first fluorophore is a fluorescent energy donor, whichis attached to a first reactant (i.e., either a nucleotide that isincorporated into the initiator or a nucleotide that is to beincorporated by the polymerase into the oligonucleotide product), and asecond fluorophore is a fluorescent energy acceptor, which is attachedto a second reactant (either a nucleotide that is incorporated into theinitiator nucleotide or a nucleotide that is to be incorporated by thepolymerase into the oligonucleotide product) that is different from thefirst reactant. In one embodiment, each of the four NTPs or NTP analogsthat may be used to extend the primer is tagged with a uniquefluorescent energy acceptor which is capable of a distinct emissionwavelength when brought into proximity with the fluorescent energy donoron the primer. Preferably, the fluorescent energy transfer can bemeasured in real time, without isolation of the oligonucleotideproducts, since neither the initiator nor unincorporated NTPs or NTPanalogs alone will produce a signal at the wavelength used fordetection.

Fluorescent and chromogenic molecules and their relevant opticalproperties are amply described in the literature. See, for example,Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2ndEdition (Academic Press, New York, 1971); Griffiths, Colour andConstitution of Organic Molecules (Academic Press, New York, 1976);Bishop, ed., Indicators (Pergamon Press, Oxford, 1972); Haugland,Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes,Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence(Interscience Publishers, New York, 1949); and the like. Further, thereis extensive guidance in the literature for derivatizing fluorophore andquencher molecules for covalent attachment via common reactive groupsthat can be added to a nucleotide, as exemplified by the followingreferences: Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345;Khanna et al., U.S. Pat. No. 4,351,760; Costas, Hanna, et al., NucleicAcids Research 28: 1849-58 (2000); Hanna, M. et al., Nucleic AcidResearch 27: 1369-76 (1999); and Meyer and Hanna, Bioconjugate Chem 7:401-412 (1996).

In general, nucleotide labeling can be accomplished through any of alarge number of known nucleotide labeling techniques using knownlinkages, linking groups, and associated complementary functionalities.Suitable donor and acceptor moieties that can effect fluorescenceresonance energy transfer (FRET) include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate; erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B, sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phthalo cyanine; and naphthalo cyanine.

There are many linking moieties and methodologies for attachingfluorophores to nucleotides, as exemplified by the following references:Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRLPress, Oxford, 1991); Zuckerman et al., Nucleic Acids Research 15:5305-5321(1987) (3′ thiol group on oligonucleotide); Sharma et al.,Nucleic Acids Research 19: 3019 (1991) (3′ sulfhydryl); Giusti et al.,PCR Methods and Applications 2: 223-227 (1993); Fung et al., U.S. Pat.No. 4,757,141 (5′ phosphoamino group via Aminolink™ II, available fromApplied Biosystems, Foster City, Calif.); Stabinsky, U.S. Pat. No.4,739,044 (3′ aminoalkylphosphoryl group); Agrawal et al., TetrahedronLetters 31: 1543-1546 (1990) (attachment via phosphoramidate linkages);Sproat et al., Nucleic Acids Research 15: 4837 (1987) (5-mercaptogroup); Nelson et al., Nucleic Acids Research 17: 7187-7194 (1989) (3′amino group); Hanna, M., Meth Enzymology 180: 383-409 (1989); Hanna, M.,Nucleic Acids Research 21: 2073-79 (1993); Hanna, M. et al., NucleicAcid Research 27: 1369-76 (1999) (5-mercapto group); Costas, Hanna, etal., Nucleic Acids Research 28: 1849-58 (2000) (8-mercapto group); andthe like.

In accordance with the invention, detection of the oligonucleotideproducts is indicative of the presence of the target sequence.Quantitative analysis is also feasible. Direct and indirect detectionmethods (including quantitation) are well known in the art. For example,by comparing the amount of oligonucleotide products that are generatedfrom a test sample containing an unknown amount of a target nucleic acidto an amount of oligonucleotide products that were generated from areference sample that has a known quantity of a target nucleic acid, theamount of a target nucleic acid in the test sample can be determined.The reiterative abortive synthesis initiation and detection methods ofthe present invention can also be extended to the analysis of geneticsequence alterations in the target nucleic acid, as further describedbelow.

Reaction Conditions

Most transcription reaction conditions are designed for the productionof full length transcripts, although no conditions have been identifiedthat eliminate abortive transcription. Appropriate reaction media andconditions for carrying out the methods of the present invention includean aqueous buffer medium that is optimized for the particularpolymerase. In general, the buffer includes a source of monovalent ions,a source of divalent cations, and a reducing agent, which is added tomaintain sulfhydral groups in the polymerase in a reduced form. Anyconvenient source of monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, ammonium sulfate, and the like, may beemployed. The divalent cation may be magnesium, managanese, zinc, or thelike, though, typically, the cation is magnesium (Mg). Any convenientsource of magnesium cations may be employed, including MgCl₂,Mg-acetate, and the like. The amount of Mg²⁺ present in the buffer mayrange from about 0.5 to 20 mM, preferably from about 1 to 12 mM.

Representative buffering agents or salts that may be present in thebuffer include Tris Phosphate, Tricine, HEPES, MOPS, and the like, wherethe amount of buffering agent typically ranges from about 5 to 150 mM,usually from about 10 to 100 mM, and preferably from about 20 to 50 mM.In certain embodiments, the buffering agent is present in an amountsufficient to provide a pH ranging from about 6.0 to 9.5, preferablyranging from about 7.0 to 8.0. Other agents which may be present in thebuffer medium include chelating agents, such as EDTA, EGTA, and thelike, or other polyanionic or cationic molecules (heparins, spermidine),protein carriers (BSA) or other proteins, including transcriptionfactors (sigma, NusA, Rho, lysozyme, GreA, GreB, NusG, etc.).

Variations in all of the reaction components potentially can alter theratio of abortive transcripts to full-length transcripts. Alterations inthe concentration of salts (from 10 mM to 100 mM) or the use ofalternative monovalent cations (K⁺ versus Na⁺ versus Rb⁺) have beenshown to affect the level of transcription (measured as abortivetranscription) on linear DNA templates (Wang, J-Y, et al., Gene196:95-98 (1997)). Alternative sulfhydral reducing reagents are reportedto have differential effects on abovtive transcription.2-mercaptoethanol at 1-2 mM is reported to enhance abortivetranscription on a poly[dA-dT] template compared to the alternativereducing agent 5,5′-dithio-bis-(2-nitrobenzoic) acid (Job, D., ActaBiochem. Pol. 41:415-419 ((1994)).

A high molar ratio of RNA polymerase to template enhances the frequencyof abortive transcription over full length transcription on the lambdaP_(R) promoter. This effect apparently arises from collisions betweentandem polymerases at the promoter.

Certain RNA polymerase mutants have elevated rates of abortivetranscription compared to the wild-type polymerase. For example, amutation changing an arginine to a cysteine at codon 529 in the RNApolymerase beta subunit gene causes elevated abortive transcriptioin atthe E. coli pyrB1 promoter (Jin, D. J. and Turnbough, Jr., C. L., J.Mol. Biol. 236:72-80 (1994)).

The relative level of abortive transcription is sensitive to thenucleotide sequence of the promoter. A number of promoters have beenidentified that are unusually susceptible to abortive transcription(e.g., the galP2 promoter). The assay system that relies on recruitmentof a defined promoter can be optimized by screening candidate promotersfor maximal initiation frequency and maximal proportion of abortivetranscripts.

Any aspect of the methods of the present invention can occur at the sameor varying temperatures. In one embodiment, the reactions are performedisothermally, which avoids the cumbersome thermocycling process. Thesynthesis reaction is carried out at a temperature that permitshybridization of the various oligonucleotides, including target siteprobes, capture probes, and APCs, as well as the primers to the targetnucleic acid template and that does not substantially inhibit theactivity of the enzymes employed. The temperature can be in the range ofabout 25° C. to about 85° C., more preferably about 30° C. to about 75°C., and most preferably about 25° C. to about 55° C. In someembodiments, the temperature for the transcription or replication stepsmay be different than the temperature(s) for the preceding steps. Thetemperature of the transcription or replication steps can be in therange of about 25° C. to about 85° C., more preferably about 30° C. toabout 75° C., and most preferably about 25° C. to about 55° C.

Denaturation of the target nucleic acid in a test sample may benecessary to carry out the assays of the present invention in caseswhere the target nucleic acid is found in a double-stranded form or hasa propensity to maintain a rigid structure. Denaturation is a processthat produces a single-stranded nucleic acid and can be accomplished byseveral methods that are well-known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (New York, Cold SpringHarbor Laboratory Press, third edition, 2000). One method for achievingdenaturation includes the use of heat, such as exposing the nucleic acidin a test sample to temperatures of about 90-100° C. for about 2-20minutes. Alternatively, a base may be used as a denaturant when thenucleic acid comprises DNA. Many basic solutions, which are well-knownin the art, may be used to denature a DNA sample. An exemplary methodincubates the DNA sample with a base, such as NaOH for example, at aconcentration of about 0.1 to 2.0 N NaOH at a temperature ranging fromabout 20° C. to about 100° C. for about 5-120 minutes. Treatment with abase, such as sodium hydroxide, not only reduces the viscosity of thesample, which increases the kinetics of subsequent enzymatic reactions,but also aids in homogenizing the sample and reducing the possibility ofbackground by destroying any existing DNA-RNA or RNA-RNA hybrids thatmay exist in the sample.

In accordance with various aspects and embodiments of the invention, thetarget nucleic acid molecules may be hybridized to an oligonucleotidecapture probe, a mononucleotide or oligonucleotide initiator which iscomplementary to a portion of the target nucleic acid, an APC linkersequence that is complementary to a portion of a target nucleic acid,and/or a target site probe that is complementary to regions on eitherside of the target site. Hybridization is conducted under standardhybridization conditions that are well-known to those skilled in theart. Reaction conditions for hybridization of an oligonucleotide (orpolynucleotide) to a target sequence vary from oligonucleotide tooligonucleotide, depending upon factors such as oligonucleotide length,the number of G:C base pairs present in the oligonucleotide, and thecomposition of the buffer utilized in the hybridization reaction.Moderately stringent hybridization conditions are generally understoodby those skilled in the art to be conditions that are approximately 25°C. below the melting temperature of a perfectly base-paireddouble-stranded DNA. Higher specificity is generally achieved byemploying more stringent conditions, such as incubation conditionshaving higher temperatures. Chapter 11 of the well-known laboratorymanual of Sambrook et al., Molecular Cloning: A Laboratory Manual (NewYork, Cold Spring Harbor Laboratory Press, 1989) describes hybridizationconditions for oligonucleotide probes and primers in great detail,including a description of the factors involved and the level ofstringency necessary to achieve hybridization with the desired degree ofspecificity.

The oligonucleotide capture probe, the target site probe, the APC,and/or the initiator may each be incubated with the target nucleic acidfor about 5 to 120 minutes at about 20 to 80° C. to permithybridization. Preferably, the target nucleic acid and theoligonucleotide probes, the APC, and/or the initiator are incubated forabout 5 to 60 minutes at about 25 to 70° C. More preferably, the targetnucleic acid and the oligonucleotide probes, the APC, and/or primer areincubated for about 5-30 minutes at about 35-50° C.

Hybridization is typically performed in a buffered aqueous solution andtemperature conditions, salt concentration, and pH are selected toprovide sufficient stringency to enable the oligonucleotide probes, theAPC, or the primer to hybridize specifically to the target sequence butnot to any other sequence. Generally, the efficiency of hybridizationbetween an oligonucleotide or polynucleotide and a target nucleic acidtemplate will be improved under conditions where the amount ofoligonucleotide or polynucleotide added to the reaction mixture is inmolar excess to the template, preferably a molar excess that ranges fromabout 10³ to 10⁶. It will be appreciated, however, that the amount oftarget nucleic acid in the test sample may not be known, so that theamount of an oligonucleotide, such as the amount of an oligonucleotidecapture probe, a target site probe, or an APC for example, relative toan amount of a target nucleic acid template cannot be determined withcertainty.

Alternatively, if a target DNA sequence has been treated with a base toeffect denaturation, the oligonucleotide or polynucleotide is diluted ina probe diluent that also acts as a neutralizing hybridization buffer.In this manner, the pH of the test sample can be kept between about 6and 9, which will favor the hybridization reaction and will notinterfere with subsequent enzymatic reactions. Preferably, theneutralizing buffer is a 2-[bis(2-hydroxyethyl) amino] ethane sulfonicacid (“BES”) (Sigma, St. Louis, Mo.) and sodium acetate buffer. Morepreferably, the neutralizing hybridization buffer is a mixture of 2 MBES, 1 M sodium acetate, 0.05% of an antimicrobial agent, such as NaN₃,5 mM of a chelating agent, such as EDTA, 0.4% of a detergent, such asTween-20™, and 20% of a hybridization accelerator, such as dextransulfate. The pH of the neutralizing hybridization buffer is betweenabout 5 to 5.5.

Transcription conditions and reagents are well-known in the art.Examples of typical conditions and reagents for RNA polymerasetranscription and DNA polymerase replication are readily found in theliterature. See, e.g., Chamberlain et al., The Enzymes, Boyer, ed., NewYork Acad. Press, 3rd ed., p. 85 (1982); Dunn et al., M. Mol. Biol. 166:477-535 (1983)); Geider, Proc. Natl. Acad. Sci. USA 75: 645-649 (1978));Guruvich et al., Analytical Biochem 195: 207-213 (1991); Lewis et al.,J. Biol. Chem. 255: 4928-4936 (1980); Martin et al., Biochem. 27:3966-3974 (1988); and Milligan et al., Methods Enzymol. Vol. 180a, ed.,50-52 (1989)). As described in Lu et al., U.S. Pat. No. 5,571,669,polymerase concentrations for transcription initiated from artificialtranscription bubble complexes are generally about one order ofmagnitude higher than the ideal polymerase concentrations forpromoter-initiated, or palindromic sequence-initiated, transcription.

In one embodiment, the foregoing components are added simultaneously atthe initiation of the abortive synthesis and detection methods. Inanother embodiment, components are added in any order prior to or afterappropriate timepoints during the method, as required and/or permittedby the various reaction steps. Such timepoints can be readily identifiedby a person of skill in the art. The enzymes used for nucleic aciddetection according to the methods of the present invention can be addedto the reaction mixture prior to or following the nucleic aciddenaturation step, prior to or following hybridization of the primer tothe target nucleic acid, prior to or following the optionalhybridization of the target site probe to the target nucleic acid, orprior to or following the optional hybridization of the APC, asdetermined by the enzymes' thermal stability and/or other considerationsknown to those skilled in the art.

The various reaction steps in the methods of the invention can bestopped at various timepoints and then resumed at a later time. Thesetimepoints can be readily identified by a person of skill in the art.Methods for stopping the reactions are known in the art, including, forexample, cooling the reaction mixture to a temperature that inhibitsenzyme activity. Methods for resuming the reactions are also known inthe art, including, for example, raising the temperature of the reactionmixture to a temperature that permits enzyme activity. In someembodiments, one or more of the components of the various reactions maybe replenished prior to, at the time of, or following the resumption ofthe reactions. Alternatively, the reaction can be allowed to proceed(i.e., from start to finish) without interruption.

Abortive Synthesis and Detection Methods of the Invention

The following examples of the abortive synthesis and detection methodsof the invention are provided to more specifically describe theinvention. These exemplary methods are intended to be merelyillustrative and are not intended to limit the description providedabove. It will be appreciated that various other embodiments may bepracticed, given the above general description. For example, referenceto the use of a primer means that any of the primers described hereinmay be used, including RNA initiators.

In accordance with an aspect of the invention, a method for detectingthe presence of a target polynucleotide by generating multipledetectable oligonucleotide products through reiterative synthesisinitiation events on the target polynucleotide is provided. FIG. 2diagrammatically illustrates the various reactants that may be combinedand reacted in the presence of RNA polymerase to synthesize multipledetectable oligonucleotide products. The methods of the invention may beperformed using a test sample that potentially contains a targetsequence. The test sequence may be detected directly or the product ofprimer-extension or reverse transcription of the target may be detected.Sequences or tags may be added to the copy of the target (e.g., biotin,ssDNA regions). The test sample may include double-stranded DNA,single-stranded DNA, or RNA. The DNA or RNA may be isolated and purifiedby standard techniques for isolating DNA or RNA from cellular, tissue,or other samples. Such standard methods may be found in references suchas Sambrook et al., Molecular Cloning: A Laboratory Manual (New York,Cold Spring Harbor Laboratory Press, third edition, 2000). In oneembodiment, the target nucleic acid is DNA or RNA that is in a suitablemedium, although the target nucleic acid can be in lyophilized form.Suitable media include, but are not limited to, aqueous media (such aspure water or buffers). In another embodiment, the target nucleic acidis immobilized prior to being utilized as a substrate for a synthesisreaction.

In an exemplary embodiment, the target sequence is immobilized by asequence-specific (e.g., gene-specific) oligonucleotide capture probethat is attached to a solid matrix, such as a microtiter plate. Theimmobilized capture probe is treated under hybridizing conditions with atest sample that includes single-stranded DNA (i.e., denatured DNA) orRNA. Any target sequence that is present in the test sample hybridizesto the capture probe and is then exposed to additional reagents inaccordance with the invention.

In an exemplary embodiment, an initiator (n 5′-R₁—(N_(I))_(x)—OH 3′)hybridizes with the target sequence upstream of a target site in thepresence of the target site probe (FIG. 11) and facilitates catalysis ofa polymerization reaction at the target site by the polymerase. Theinitiation primer may be comprised of nucleosides, nucleoside analogs,nucleotides, and nucleotide analogs. The initiaor primer may vary in thenumber of nucleotides, such as nucleotides from 1-25 nucleotides, 26-50nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides,126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225nucleotides, 226-250 nucleotides, and greater than 250 nucleotides, andmay include one or more nucleotide analogs. A suitable RNA polymerase isemployed to synthesize an oligoribonucleotide product from the targetsequence or any portion thereof. The polymerase may be an RNA-dependentor DNA-dependent RNA polymerase. The DNA or RNA target sequence may ormay not be attached to other molecules, such as proteins, for example.

During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotideswhich have been added to the reaction mixture. As the polymerasereaction proceeds, the polymerase extends the initiator, as directed bythe template sequence, by incorporating corresponding nucleotides thatare present in the reaction mixture. In one embodiment, these reactantnucleotides comprise a chain terminator (e.g., n 5′ pppN_(T)—R₂, a chainterminating nucleotide analog, as described above). When the polymeraseincorporates a chain terminator into the nascent oligonucleotideproduct, chain elongation terminates due to the polymerase's inabilityto catalyze the addition of a nucleotide at the 3′ position on thepentose ring of the chain terminator. Consequently, the polymeraseaborts the initiated synthesis event by releasing the oligonucleotideproduct (i.e., 5′ R₁—(N_(I))_(z)pN_(T)—R₂, where z=x+y) and reinitiatingthe abortive initiation synthesis reaction at the target site.

The abortive initiation reaction may be controlled such that thepolymerase aborts synthesis after extending the initiator by apredetermined number of nucleotides. For example, if it is desirable toterminate the synthesis reaction after the initiator has been extendedby a single nucleotide, this may be accomplished by, for example,either: (1) adding to the reaction mixture only nucleotides that arechain terminators, thereby inhibiting polymerization after the firstnucleotide is incorporated by the polymerase; or (2) if the geneticsequence of the target site is known, adding to the reaction mixtureonly a preselected chain terminating nucleotide analog (i.e., nucleotideanalogs which comprise one of A, G, T, C, or U) that is complementary tothe nucleotide at the target site. Alternatively, if it is desirable toterminate the synthesis reaction after the initiator has been elongatedby a predetermined number of nucleotides, and if the genetic sequence ofthe target site is known, this may be accomplished by, for example,adding to the reaction mixture a preselected chain terminatingnucleotide analog (i.e., nucleotide analogs which comprise one of A, G,T, C, or U) that is complementary to an Nth nucleotide from the targetsite, where N is the predetermined number of nucleotides comprised bythe oligonucleotide product, exclusive of the initiator. In this manner,multiple abortive oligonucleotide products that comprise the initiatorand a chain terminating nucleotide analog are synthesized by thepolymerase.

The polymerase releases the oligonucleotide product withouttranslocating from the enzyme binding site or dissociating from thetarget polynucleotide sequence. Nucleotide deprivation can be used tosequester the polymerase at the polymerase binding site. For example, ifonly an initiator and a terminator are supplied, elongation by thepolymerase will not be possible.

Furthermore, reaction conditions may be optimized for abortivetranscription initiation, whereby it is favorable for the polymerase toremain bound to the polymerase binding site even in the presence ofelongating nucleotides. The abortive initiation reaction buffer will beoptimized to increase the abortive events by adjusting theconcentrations of the salts, the divalent cations, the glycerol content,and the amount and type of reducing agent to be used. In addition,“roadblock” proteins may be used to prevent the polymerase fromtranslocating.

In another aspect of the invention, the initiator includes a moiety(e.g., R₁, as depicted in FIG. 2) which may be covalently bonded to the5′ phosphate group (as in FIG. 3), the 2′ position of the pentose ring,or the purine or pyrimidine base of one of the nucleotides or nucleotideanalogs that are incorporated into the initiator. Additionally, thereactant nucleotides and/or nucleotide analogs that are included in thereaction mixture for incorporation into the oligonucleotide product bythe polymerase may each also include a moiety (e.g., R₂, as depicted inFIG. 2), which is covalently bonded to either the nucleobase (as in FIG.4) or the 2′ position or 3′ position of the pentose ring. In anexemplary embodiment, R₁ and R₂ are label moieties (as in FIG. 5) on theinitiator and the chain terminator, respectively, that are incorporatedinto the oligonucleotide product by the polymerase (as in FIG. 6) andare adapted to interact in a manner that generates a detectable signal(e.g., fluorescence resonance energy transfer (FRET) (FIG. 7),fluorescence or colorimetry (FIG. 8)), thereby permitting the detectionand quantitation of the synthesized oligonucleotide products. In oneembodiment, as illustrated in FIG. 9, an oligonucleotide product (5′ R₁—(N_(I))_(x)pN_(T)—R₂) incorporating an initiator (N_(I)) that has anenergy donor group (R₁) and a chain terminating nucleotide (N_(T)) thathas an energy acceptor group (R₂) generates a signal throughfluorescence resonance energy transfer from R₁ to R₂ when thesynthesized oligonucleotide products are irradiated with light of aparticular wavelength. As shown in FIG. 9, when the energy donor moietyR₁ on the initiator is excited by exposure to light of a specifiedwavelength (λ_(1A)) (e.g., the absorption maximum of R₁) the exciteddonor moiety R₁ emits light of a second wavelength (λ_(1E/2A)) (e.g.,the emission maximum for R₁) which is absorbable by R₂. If N_(T) hasbeen suitably incorporated into the oligonucleotide product by thepolymerase, the energy acceptor moiety R₂ on N_(T) is positionedsufficiently near R₁ on N_(I) (e.g., within about 80 Å) to facilitateefficient energy transfer between R₁ and R₂, such that R₂ absorbs thewavelength of light (λ_(1E/2A)) emitted by the excited donor moiety R₁.In response to the absorption of λ_(1E/2A), the excited R₂ acceptormoiety emits light of a third wavelength (λ_(2E)), which may then bedetected and quantified in accordance with methods that are well-knownin the art. Exemplary R₁ and/or R₂ FRET label moieties include aedansand fluorescein (as shown in FIG. 7), or pyrene, stilbene, coumarine,bimane, naphthalene, pyridyloxazole, naphthalimide, NBD, BODIPY™, aswell as any of those described in greater detail above.

In an alternate embodiment, as diagrammatically illustrated in FIG. 10,n copies of a dinucleotide initiator (5′ R₁—N₁pN₂—R₂—OH 3′) comprisingreporter moieties (R₁ and R₂) on either of the nucleotides (N₁ and N₂,respectively) may be extended by a polymerase to incorporate n copies ofa chain terminator (5′ pppN₃R₃) which includes a third reporter moiety(R₃), yielding n copies of a detectable trinucleotide transcript (5′R₁—N₁pN₂R₂pN₃—R₃—OH 3′). In a manner similar to the one described abovewith reference to FIG. 10, the trinucleotide transcript may beirradiated with a first wavelength of light (λ_(1A)) which excites theR₁ energy donor group on the first nucleotide (N₁) to emitλ_(1E)/λ_(3A). λ_(1E)/λ_(3A) is then absorbed by the R₃ energy acceptorgroup on the chain terminating nucleotide (N₃), and an excited R₃ thenemits λ_(3E), which can then be detected and quantified. Alternatively,the transcript may be irradiated with a second wavelength of light(λ_(2A)) which excites an R₂ energy donor group on a second nucleotide(N₂) to emit λ_(2E)/λ_(3A). λ_(2E)/λ_(3A) is then absorbed by the R₃energy acceptor group, and an excited R₃ then emits λ_(3E), which can bedetected and quantified. In either case, the detectable wavelength(λ_(3E)) is not obtained unless the polymerase brings an energy donorreporter moiety on the initiator (R₁ or R₂) into sufficient proximitywith a corresponding energy acceptor reporter moiety (R₃) on theincorporated nucleotide to result in the emission of the detectablewavelength of light.

In another aspect of the invention, as diagrammatically illustrated inFIG. 11, a target site probe may be used to form a bubble complex in atarget region of the target sequence. As described above, the bubblecomplex comprises double-stranded regions that flank a single-strandedregion that includes a target site. In this embodiment, the target siteprobe is used to direct the polymerase to the target site by positioningthe target site at the junction of the single-stranded bubble region anda downstream duplex region on the target sequence. In an exemplaryembodiment, the target site probe comprises from about 18-54nucleotides: a first region (A) which hybridizes to the target sequence(A′) upstream of the target site comprises about 5-20 nucleotides; aninternal, second region of non-base-paired nucleotides (B) comprisesabout 8-14 nucleotides; and a third region (C) which hybridizes to thetarget sequence downstream of the target site (C′) comprises about 5-20nucleotides. The polymerase associates with an initiator and initiates asynthesis reaction at the target site on the template sequence. Thepolymerase elongates the initiator to synthesize an abortiveoligonucleotide product through the incorporation of nucleotides, whichcomprise a suitable chain terminator. Both the initiator and thenucleotides, including the chain terminating nucleotide, may be modifiedwith a label moiety to allow signal detection, such as by fluorescenceresonance energy transfer for example, as described above.

An illustrative procedure for detecting multiple oligonucleotideproducts through reiterative synthesis initiation events on a targetsequence, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isdesigned to hybridize with a specific or general target sequence; (b)optionally hybridizing the oligonucleotide capture probe with a testsample which potentially contains a target sequence; (c) optionallyhybridizing the target sequence with a target site probe; (d) modifyingat least one of an initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide productsynthesized by the polymerase; (e) hybridizing the target sequence withthe primer; and (f) extending the initiator with a polymerase such thatthe polymerase reiteratively synthesizes an oligonucleotide product thatis complementary to a target site by incorporating complementarynucleotides comprising a chain terminator and releasing an abortiveoligonucleotide product without either translocating from an enzymebinding site or dissociating from the target sequence.

During transcription of the template by the RNA polymerase, the RNAinitiator is extended by the RNA polymerase through the incorporation ofnucleotides that have been added to the reaction mixture. As thepolymerase reaction proceeds, the RNA polymerase extends the RNAinitiator, as directed by the template sequence, by incorporatingcorresponding nucleotides that are present in the reaction mixture. Inone embodiment, these reactant nucleotides comprise a chain terminator(e.g., n 5′ pppN_(T)—R₂, a chain terminating nucleotide analog, asdescribed above). When the RNA polymerase incorporates a chainterminator into the nascent transcript, chain elongation terminates dueto the polymerase's inability to catalyze the addition of a nucleotideat the 3′ position on the ribose ring of the chain terminator, and theRNA polymerase aborts the initiated transcription event by releasing thetranscript and reinitiating transcription at the target site. Theabortive transcription initiation reaction may be controlled such thatmultiple abortive oligonucleotide transcripts of a predetermined lengthand comprising the RNA primer and a chain terminating nucleotide analogare generated.

In an exemplary embodiment, the RNA initiator may be a mononucleotideand the nucleotides provided in the reaction mixture may comprise solelychain terminators. In this embodiment, transcription is aborted by theRNA polymerase after the RNA initiator has been extended by a singlenucleotide and an abortive dinucleotide transcript is generated. Inanother embodiment, the RNA initiator may comprise a dinucleotide or atrinucleotide, for example, and an abortive transcription initiationevent may generate an abortive transcript comprising a trinucleotide ora tetranucleotide, respectively. It will be appreciated that abortivetranscripts of any desired length may be obtained, depending upon thelength of the RNA initiator and the nature and composition of thereactant nucleotides that are selected for inclusion in the reactionmixture. For example, if the nucleotide sequence of the template isknown, the components (e.g., target site, initiator, and reactantnucleotides) of the transcription reaction may be selected such thatabortive transcripts of any desired length are generated by the methodof the invention.

In another aspect of the invention, the RNA initiator includes a moiety(e.g., R₁, as depicted in FIG. 6) which may be covalently bonded to the5′ phosphate group, the 2′ position of the ribose ring, or the purine orpyrimidine base of one of the nucleotides or nucleotide analogs that areincorporated into the RNA initiator. Additionally, the reactantnucleotides and/or nucleotide analogs that are included in the reactionmixture for incorporation into the oligonucleotide transcript by the RNApolymerase may each also include a moiety (e.g., R₂, as depicted in FIG.6), which is covalently bonded to either the nucleobase or the 2′position or 3′ position of the ribose ring. The moieties R₁ and R₂ mayeach comprise H, OH, or any suitable label moiety, reporter group, orreporter group precursor, as described in greater detail above.

An illustrative procedure for detecting multiple oligonucleotidetranscripts through reiterative transcription initiation events on atarget sequence, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isdesigned to hybridize with a specific or general target sequence; (b)optionally hybridizing the oligonucleotide capture probe with a testsample which potentially contains a target sequence; (c) optionallyhybridizing the target sequence with a target site probe; (d) modifyingat least one of an RNA initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide transcriptsynthesized by the RNA polymerase; (e) hybridizing the target sequencewith the RNA initiator; and (f) extending the RNA initiator with an RNApolymerase such that the RNA polymerase reiteratively synthesizes anoligonucleotide transcript that is complementary to a target site byincorporating complementary nucleotides comprising a chain terminatorand releasing an abortive oligonucleotide transcript withoutsubstantially translocating from the polymerase binding site ordissociating from the target sequence.

In accordance with another aspect of the invention, as diagrammaticallyillustrated in FIG. 8, the methods of the invention may be utilized togenerate an oligonucleotide product (5′ R₁—(N_(I))_(x)pN_(T)—R₂) whichcomprises an initiator (N_(I)) with a moiety (R₁), such as animmobilization tag for example; and a chain terminating nucleotide(N_(T)) that includes a label moiety (R₂), such as a signal generator orsignal generator precursor for example. In this embodiment, theoligonucleotide product(s) may be captured or immobilized, such as on amembrane for example, to facilitate detection of the oligonucleotideproducts of the abortive synthesis reaction. In an exemplary embodiment,R₁ is a bioadhesive tag, such as biotin for example; R₂ is a labelmoiety, such as fluorescein for example; and oligonucleotide productsthat are attached to the solid matrix by the R₁ bioadhesive tag arecapable of direct detection through an emission from the R₂ labelmoiety. In another exemplary embodiment, an antibody, such asanti-dinitrophenyl (anti-DNP) for example, is attached to the solidmatrix; R₁ is an immobilization tag, such as dinitrophenyl (DNP) forexample; R₂ is a reporter or reporter precursor, such as a reactivethiol for example; and, upon silver/gold development, theoligonucleotide products that are attached to the solid matrix by the R₁tag produce a colored signal that is visible to the naked eye withoutirradiation.

Applications of the Abortive Synthesis and Detection Methods of theInvention

The methods of the present invention can be used in a variety ofdiagnostic contexts. For purposes of illustration, methods of assessingthe methylation state of specific genes, detecting the presence of knowngenetic mutations, detecting the presence of pathogenic organisms,detecting mRNA expression levels, and detecting and amplifying proteinsare described.

DNA Methylation

The methods of the present invention-may be used in diagnostic assayswhich detect epigenetic changes associated with disease initiation andprogression by assessing the methylation state of specific genes andtheir regulatory regions that are known to be associated with particulardisease-states. DNA methylation is a cellular mechanism for altering theproperties of DNA without altering the coding function of that sequence.The methylation reaction, which is catalyzed byDNA-(cystosine-5)-methyltransferase, involves the transfer of a methylgroup from S-adenosylmethionine to the target cytosine residue to form5-methylcytosine (5-mCyt) (FIG. 12). See Gonzalgo et al., U.S. Pat. No.6,251,594. The areas of the genome that contain 5-mCyt at CpGdinucleotides are referred to as “CpG islands.” While changes in themethylation status of the cytosine residues in DNA CpG islands commonlyoccur in aging cells, altered gene methylation (either increased ordecreased) is frequently an early and permanent event in many types ofdisease, including cancer. CpG islands tend to be found in DNAregulatory regions that are near genes and determine whether these genesare either active or inactive. Many genes that regulate cell growth, andtherefore prevent or inhibit the development of cancer, such as tumorsuppressor genes, must be active (unmethylated) to promote normal cellgrowth. Other genes, such as oncogenes for example, must be inactive(methylated) so as not to promote abnormal cell growth.

For example, many types of cancer are associated with a distinctcombination or pattern of CpG island methylation. FIG. 16 graphicallyillustrates the manner in which altered gene methylation may beassociated with various types of cancer. The graph plots 13 exemplarycancers (prostate, kidney, bladder, esophageal, lung, gastric, colon,blood, breast, skin, brain, liver, and ovarian) against 49 genes whichhave been shown to have methylation changes that are associated with theinitiation and progression of the identified types of cancer. Each ovalin the graph (coded by cancer type) indicates an abnormal methylationstatus for a gene (i.e., methylated when its normal status isunmethylated or unmethylated when its normal status is methylated).Since each type of cancer may be associated with a different pattern ofmethylation-altered genes, cancer-affected organs may potentially beidentified based upon organ-specific combinations of methylated genes.For example, in the case of prostate cancer cells, genes 4, 9, 10, 14,19, 22, 32, and 33 have been shown to exhibit abnormal methylationstates. Thus, if standardized diagnostics could easily evaluate themethylation states of these 8 genes, then the initiation, progression,and recurrence of prostate cancer could be readily monitored and moreeffective patient treatment strategies could be developed. It will beappreciated that FIG. 16 represents only a subset of the genes for whichaltered methylation states and patterns are indicative of various typesof cancer.

In an exemplary embodiment, the methods of the invention may be utilizedto monitor disease initiation, progression, metastasis, recurrence, andany responses to treatment therapies by providing diagnostic techniques,which can detect altered methylation states and patterns. Methylatedcytosine residues in a DNA fragment can be detected based upon theresistance of such residues to deamination by a deaminating agent, suchas sodium bisulfite for example. When denatured (i.e., single-stranded)DNA is exposed to a deaminating agent, such as sodium bisulfite,unmethylated cytosine (C) residues are converted into uracil residues(U), while methylated cytosine residues (5-mCyt) remain unchanged. Thatis, as illustrated in FIG. 14, deamination resulting from a treatmentwith sodium bisulfite causes the originally unmethylated cytosines tochange their complementary base-pairing partner from guanine (G) toadenosine (A). However, the methylated cytosines (5-mCyt) retain theirbase-pairing specificity for G. Thus, after deamination by sodiumbisulfite, a target DNA sequence will have only as many complementaryCpG islands as there were methylated CpG islands in the original,untreated target DNA sequence. Additionally, as further illustrated inFIG. 14, if an original, untreated target DNA sequence has no methylatedCpG islands, then the bisulfite-treated target DNA sequence will nolonger contain any CpG islands.

In view of the foregoing, the level of methylation of the CpG islands ina target DNA sequence may be determined by measuring the relative levelof unaltered CpG sites. This relative measurement may be accomplished byinitiating abortive transcription at the CpG sites that remain after thetarget DNA sequence has been exposed to a deaminating agent, such assodium bisulfite. The sodium bisulfite reaction is performed accordingto standard techniques. See, e.g., Gonzalgo et al., U.S. Pat. No.6,251,594. In one embodiment, as illustrated in FIG. 15, a sodiumbisulfite-treated DNA target sequence can be incubated with an RNApolymerase and an initiator, such as a mononucleotide initiator (5′R₁—C—OH 3′) for example. The initiator associates with the polymeraseand initiates transcription and RNA synthesis at an intact CpG site onthe DNA template. Each CpG site can direct the extension of an initiatorto synthesize an abortive transcript (e.g., 5′ R₁-CpG-R₂ 3′) through theincorporation of a suitable chain terminator (e.g., pppG-R₂), asillustrated at Sites 1, 3, and 4 in FIG. 15. Either or both of theinitiator and a chain terminating nucleotide may be modified with alabel moiety (e.g., R₁ and R₂, respectively) to allow signal detection.In an exemplary embodiment, the transcripts may be detected throughfluorescence resonance energy transfer (FRET) for example, as describedin detail above (e.g., the primer contains an energy donor (R₁) at its5′-end, and the NTP contains an energy acceptor (R₂) attached to thenucleobase).

In an alternate embodiment, a sodium bisulfite-treated DNA targetsequence may be incubated with an RNA polymerase and a dinucleotideinitiator (5′ R₁-CpG-OH 3′). The initiator then associates with thepolymerase and initiates transcription and RNA synthesis at an intactCpG site on the DNA template. Each CpG site then directs the extensionof the dinucleotide initiator to synthesize an abortive trinucleotidetranscript through the incorporation of a suitable chain terminator. Thenucleotide analog that comprises the chain terminator will depend uponthe DNA template sequence. For example, at Site 1 of FIG. 15, a suitablechain terminator would include 5′ pppA-R₂ 3′, and the resultant abortivetrinucleotide transcript would be 5′ R₁-CpGpA-R₂ 3′.

In another embodiment, as diagrammatically illustrated in FIG. 15, afterthe target DNA sequence has been deaminated, such as by treating thetarget DNA sequence with sodium bisulfite for example, a target siteprobe may be used to form a bubble complex that comprises a target CpGsite on the target DNA sequence. In this embodiment, the target siteprobe is used to direct the RNA polymerase to the target CpG site bypositioning the target CpG site at the junction of a single-strandedbubble region and a downstream duplex region on the target DNA sequence.In the illustrated embodiment, the target site probe comprises about18-54 nucleotides: a first region which hybridizes to the target DNAsequence upstream of the target site comprises about 5-20 nucleotides;an internal second region of non-base-paired nucleotides comprises about8-14 nucleotides; and a third region which hybridizes to the target DNAsequence downstream of the target site comprises about 5-20 nucleotides.The target site probe may be hybridized to the target DNA sequenceeither before or while the DNA target sequence is incubated with an RNApolymerase and a suitable RNA initiator. The polymerase associates withthe RNA initiator and initiates transcription and RNA synthesis at theCpG site on the DNA template. The polymerase extends the initiator tosynthesize an abortive oligonucleotide transcript through theincorporation of a suitable chain terminator. Either or both of theinitiator and a chain terminating nucleotide may be modified with alabel moiety to allow signal detection, such as by fluorescenceresonance energy transfer for example, as described in detail above.

In another embodiment, capture probes may be designed to capture thegenes of interest, and abortive transcription initiation used todetermine the methylation status of the desired genes. For example,genes known to be associated with the progression of a particularcancer, such as colon cancer, may be monitored, including but notlimited to APC (adenomatous polyposis coli), CALCA (calcitonin), ER(estrogen receptor), GSTP1, HIC1 (hypermethylated in cancer-1), hMLH1,HPP1TR/TENB2/TMEFF2 (Transmembrane protein with EFG-like and twofollistatin-like domains 2), LKB1/STK11. IGF2 IGF2 (Insulin-like growthfactor), MGMT (O⁶ methyl guanine methyl transferase 1), MINT25,p14(ARF), p16(INK4a)/MTSI/CDKN2A, PAX6 (paired box gene 6), RAR-Beta2,THBS1 (thrombospondin-1), Veriscan, and WT1 (Wilm's tumor suppressor).Each gene of interest could be removed from the sample by hybridizationto a capture sequence, which is unique for the gene of interest. Thecapture sequence may be immobilized on a solid matrix, including but notlimited to magnetic beads, microtiter plates, sepharose, agarose, cationexchange resins, lateral flow strips, glass beads, and microarray chips.Once the gene of interest has been removed from the sample, abortivetranscription initiation can be used to determine the methylation statusfor each particular gene.

An illustrative procedure for detecting DNA methylation states andpatterns, therefore, may include the following process steps: (a)optionally immobilizing an oligonucleotide capture probe which isspecific for a region near a CpG island of a target gene; (b) optionallytreating the oligonucleotide capture probe with a denatured DNA samplewhich potentially contains a target DNA sequence; (c) converting anyunmethylated cytosine residues on the target DNA sequence to uracilresidues and leaving any methylated cytosine residues unaltered; (d)optionally hybridizing the target DNA sequence with a target site probe;(e) modifying at least one of an RNA initiator and nucleotidescomprising a chain terminator to enable detection of the oligonucleotidetranscript; (f) hybridizing the target DNA with the RNA initiator; and(g) extending the RNA initiator with an RNA polymerase such that the RNApolymerase reiteratively synthesizes an oligonucleotide transcript thatis complementary to a target site by incorporating complementarynucleotides comprising a chain terminator and releasing an abortiveoligonucleotide transcript without either translocating from an enzymebinding site or dissociating from the target DNA sequence; and (g)detecting and optionally quantifying the multiple abortiveoligonucleotide transcripts.

Genetic Mutations

In another aspect of the invention, the methods disclosed herein may beused in diagnostic assays which detect mutations in the form of grosschromosomal rearrangements or single or multiple nucleotide alterations,substitutions, insertions, or deletions. In an exemplary embodiment, asdiagrammatically illustrated in FIG. 17, single nucleotide polymorphisms(SNPs) may be detected through the use of an abortive oligonucleotidesynthesis reaction. A known target SNP sequence (e.g., 3′dN_(X′)pdN_(Y′)pdN_(T′)5′, where dN_(T′) is a target SNP site) can beincubated with an RNA polymerase, an RNA initiator, such as adinucleotide initiator for example, and nucleotides (e.g., a chainterminator such as 5′ pppN_(T)—R₂). The initiator binds immediatelyupstream of the target SNP sequence, associates with the polymerase, andinitiates transcription and RNA synthesis at the target SNP site. In oneembodiment, the polymerase elongates the initiator by incorporating thechain terminator to produce an abortive trinucleotide product. Either orboth of the initiator and a chain terminating nucleotide may be modifiedwith a label moiety (R₁ and R₂, respectively) to allow signal detection.In an exemplary embodiment, the transcripts may be detected throughfluorescence resonance energy transfer (FRET) for example, as describedin detail above (e.g., the initiator contains an energy donor (R₁) atits 5′-end, and the chain terminator contains an energy acceptor (R₂)attached to the nucleobase).

An illustrative procedure for detecting mutations in a target DNAsequence (FIG. 18), therefore, may include the following process steps:(a) optionally immobilizing a capture probe designed to hybridize with atarget DNA sequence which includes a mutation; (b) optionallyhybridizing the capture probe with a DNA sample which potentiallycontains the target DNA sequence; (c) optionally hybridizing the targetDNA sequence with a target site probe; (d) modifying at least one of anRNA initiator (R₁N₁—OH) and nucleotides comprising a chain terminator(pppN_(T)—R₂) to enable detection of the oligonucleotide transcriptsynthesized by the RNA polymerase; (e) hybridizing the target DNAsequence with the RNA initiator; (f) extending the RNA initiator with anRNA polymerase such that the RNA polymerase reiteratively synthesizes anoligonucleotide transcript that is complementary to a target mutationsite by incorporating complementary nucleotides comprising a chainterminator and releasing an abortive oligonucleotide transcript withouteither translocating from an enzyme binding site or dissociating fromthe target DNA sequence; and (g) detecting and optionally quantifyingthe multiple abortive oligonucleotide transcripts.

Pathogenic Organisms

In another aspect of the invention, the methods disclosed herein may beused in diagnostic assays which detect the presence of a particularnucleic acid (DNA or RNA), thereby serving to indicate the presence ofeither a particular or a generic organism which contains the gene, orwhich permit genetic typing of a particular organism without the needfor culturing the organism. The test sample may be suspected ofcontaining a target nucleic acid sequence from a particularmicroorganism, such as bacteria, yeast, viruses, viroids, molds, fungi,and the like. The test sample may collected from a variety of sourcesincluding but not limited to, animal, plant or human tissue, blood,saliva, semen, urine, sera, cerebral or spinal fluid, pleural fluid,lymph, sputum, fluid from breast lavage, mucusoal secretions, animalsolids, stool, cultures of microorganisms, liquid and solid food andfeedproducts, waste, cosmetics, air, and water.

In another aspect of the invention, the methods disclosed herein may beused in diagnostic assays which detect the presence of a particularnucleic acid (DNA or RNA), thereby serving to indicate the presence ofeither a particular or a generic pathogenic organism which contains thegene, or which permit genetic typing of a particular organism withoutthe need for culturing the organism. In an exemplary embodiment, asdiagrammatically illustrated in FIG. 19, an oligonucleotide captureprobe that is sequence-specific for a target pathogen polynucleotide isattached to a solid matrix, such as a microtiter plate for example, andthe capture probe is treated under hybridizing conditions with a testsample which potentially contains the target pathogen polynucleotide.The test sample may be suspected of containing a target nucleic acidsequence from a particular pathogen, such as, for example, amicroorganism, such as bacteria, yeast, viruses, viroids, molds, fungi,and the like. The test sample may collected from a variety of sourcesincluding but not limited to, animal, plant or human tissue, blood,saliva, semen, urine, sera, cerebral or spinal fluid, pleural fluid,lymph, sputum, fluid from breast lavage, mucusoal secretions, animalsolids, stool, cultures of microorganisms, liquid and solid food andfeedproducts, waste, cosmetics, air, and water.

The target pathogen polynucleotide may be either RNA or DNA. A targetpathogen polynucleotide that is present in the test sample hybridizes tothe capture probe, and a washing step is then performed to remove anycomponents of the test sample that were not immobilized by the captureprobe. Target DNA or RNA may be retrieved by addition of specificsequences via primer extension, for example. In an exemplary embodiment,the captured target pathogen polynucleotide is hybridized with anabortive promoter cassette (APC). The APC linker sequence includes asingle-stranded overhang region on either its 3′ or 5′ end (dependingupon the orientation needed to create an antiparallel hybrid with thecapture probe). In other words, the APC linker is complementary to thesequence on the free end of the captured target pathogen polynucleotide,thereby permitting the APC linker to hybridize to the target pathogenpolynucleotide.

An initiator and a polymerase are added to the reaction mixture. Theinitiator hybridizes within the bubble region of the APC at a positionthat facilitates catalysis of a synthesis reaction by a suitablepolymerase at the target site. The initiator may be RNA or DNA, maycomprise from about 1 to 25 nucleotides, and may include one or morenucleotide analogs as well as nucleotides. The polymerase may be anRNA-dependent or DNA-dependent RNA polymerase. The DNA or RNA APC may ormay not be attached to other molecules, such as proteins, for example.In an exemplary embodiment, the APC comprises DNA, the initiator is RNA,and the polymerase is a DNA-dependent RNA polymerase.

During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotidesthat have been added to the reaction mixture. As the polymerase reactionproceeds, the polymerase extends the initiator, as directed by the APCtemplate sequence within the bubble region, by incorporatingcomplementary nucleotides, including a suitable chain terminator, thatare present in the reaction mixture. When the polymerase incorporates achain terminator into the nascent oligonucleotide product, chainelongation terminates due to the polymerase's inability to catalyze theaddition of a nucleotide at the 3′ position on the pentose ring of theincorporated chain terminator. Consequently, the polymerase aborts theinitiated synthesis event by releasing the oligonucleotide product andreinitiating the synthesis reaction at the target site. Either or bothof the initiator and a chain terminating nucleotide may be modified witha label moiety to allow signal detection. In an exemplary embodiment,the oligonucleotide products may be detected through fluorescenceresonance energy transfer (FRET), as described above (e.g., theinitiator contains an energy donor (R₁) at its 5′-end, and the chainterminator contains an energy acceptor (R₂) attached to the nucleobase).

An illustrative procedure for detecting the presence of pathogens (FIG.20), therefore, may include the following process steps: (a) optionallyimmobilizing a capture probe designed to hybridize with a targetpathogen polynucleotide; (b) optionally hybridizing the capture probewith a test sample which potentially contains a target pathogenpolynucleotide. The target nucleic acid may be copied to DNA via reversetranscription (for RNA pathogens) or primer extension (for DNApathogens). In both bases, a DNA sequence corresponding to the AbortivePromoter Cassette (APC) linker will be added to the target copy (FIG.1); (c) optionally washing the captured target pathogen polynucleotideto remove any unhybridized components of the test sample; (d)hybridizing the captured target pathogen polynucleotide with an abortivepromoter cassette; (e) modifying at least one of a initiator andnucleotides comprising a chain terminator to enable detection of theoligonucleotide product synthesized by the polymerase; (f) hybridizingthe abortive promoter cassette with a initiator; (g) extending theinitiator with a polymerase such that the polymerase reiterativelysynthesizes an oligonucleotide product that is complementary to a targetsite by incorporating complementary nucleotides comprising a chainterminator and releasing an abortive oligonucleotide product withouteither translocating from an enzyme binding site or dissociating fromthe APC; and (h) detecting and optionally quantifying the multipleabortive oligonucleotide products.

The present invention is useful for detecting pathogens in mammals. Inparticular the invention is useful for the detection of bacteria,viruses, fungus, molds, amoebas, prokaryotes, and eukaryotes. Preferredmammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbitsand humans. Particularly preferred are humans.

The methods of the invention are particularly useful for monitoring thepresence or absence of pathogenic nucleic acids and proteins. Theinvention can be used to detect, diagnose, and monitor diseases, and/ordisorders associated with pathogenic polypeptides or polynucleotides.The invention provides for the detection of the aberrant expression of apolypeptide or polynucleotide. The method comprises (a) assaying theexpression of the polypeptide or polynucleotide of interest in cells,tissue or body fluid of an individual using the methods of abortiveinitiation transcription described above, and (b) comparing the level ofgene expression, protein expression, or presence of sequences ofinterest with a standard gene or protein expression level or seqeunce ofinterest, whereby an increase or decrease in the assayed polypeptide orpolynucleotide level compared to the standard level is indicative ofaberrant expression indicating presence of a pathogen of interest.

The presence of an abnormal amount of transcript in biopsied tissue orbody fluid from an individual may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the disease causedby the pathogen.

The invention is particularly useful for monitoring the presence ofpathogenic organisms including but not limited to E. coli, Steptococcus,Bacillus, Mycobacterium, HIV, and Hepatitis.

The methods of the invention may be used to test for pathogenicmicroorganisms in aqueous fluids, in particular water (such as drinkingwater or swimming or bathing water), or other aqueous solutions (such asfermentation broths and solutions used in cell culture), or gases andmixtures of gases such as breathable air, and gases used to sparge,purge, or remove particulate matter from surfaces. Breathable air fromany source including but not limited to homes, schools, classrooms,workplaces, aircraft, spacecraft, cars, trains, buses, and any otherbuilding or structure where people gather, may be tested for thepresence of pathogenic microorganisms.

mRNA Expression

In another aspect of the invention, the methods disclosed herein may beused in diagnostic assays which detect messenger RNA (mRNA) expressionlevels in a quantitative or non-quantitative manner. In an exemplaryembodiment, as diagrammatically illustrated in FIG. 21, anoligonucleotide capture probe that is sequence-specific for a targetmRNA sequence is attached to a solid matrix, such as a microtiter platefor example, and the capture probe is treated under hybridizingconditions with a test sample which is suspected of containing thetarget mRNA sequence. A target mRNA sequence that is present in the testsample hybridizes to the capture probe, and a washing step is thenperformed to remove any components of the test sample that were notimmobilized by the capture probe. The captured target mRNA sequence isthen hybridized with an abortive promoter cassette (APC). In theillustrated embodiment, the APC has an APC linker sequence whichincludes a single-stranded poly-T overhang on its 3′ end that iscomplementary to the poly-A tail on the 3′ end of the target mRNAsequence, thereby permitting the APC linker to hybridize to the poly-Atail of the target mRNA.

An initiator and a polymerase are added to the reaction mixture. Theinitiator hybridizes within the bubble region of the APC, upstream ofthe target site, and facilitates catalysis of a synthesis reaction by asuitable polymerase at the target site. The initiator may comprise fromabout 1 to 25 nucleotides, and may include one or more nucleotideanalogs as well as nucleotides. The polymerase may be an RNA-dependentor DNA-dependent RNA polymerase. The APC may or may not be attached toother molecules, such as proteins, for example. In an exemplaryembodiment, the APC comprises DNA, the initiator is RNA, and thepolymerase is a DNA-dependent RNA polymerase.

During the polymerization reaction, the initiator is extended orelongated by the polymerase through the incorporation of nucleotideswhich have been added to the reaction mixture. As the polymerasereaction proceeds, the polymerase extends the initiator, as directed bythe APC template sequence within the bubble region, by incorporatingcomplementary nucleotides, including a chain terminator, that arepresent in the reaction mixture. When the polymerase incorporates achain terminator into the nascent oligonucleotide product, chainelongation terminates due to the polymerase's inability to catalyze theaddition of a nucleotide at the 3′ position on the pentose ring of theincorporated chain terminator. Consequently, the polymerase aborts theinitiated synthesis event by releasing the oligonucleotide product andreinitiating the synthesis reaction at the target site. Either or bothof the initiator and a chain terminating nucleotide may be modified witha label moiety to allow signal detection, such as by fluorescenceresonance energy transfer for example, as described in detail above.

An illustrative procedure for detecting mRNA expression levels,therefore, may include the following process steps: (a) optionallyimmobilizing a capture probe designed to hybridize with a specific orgeneral mRNA sequence; (b) optionally hybridizing the capture probe witha test sample which potentially contains a target mRNA sequence; (c)optionally washing the captured target mRNA sequence to remove anyunhybridized components of the test sample; (d) hybridizing the capturedtarget mRNA sequence with an abortive promoter cassette; (e) modifyingat least one of a initiator and nucleotides comprising a chainterminator to enable detection of the oligonucleotide productsynthesized by the polymerase; (f) hybridizing the abortive promotercassette with the initiator; (g) extending the initiator with apolymerase such that the polymerase reiteratively synthesizes anoligonucleotide product that is complementary to a target site byincorporating complementary nucleotides comprising a chain terminatorand releasing an abortive oligonucleotide product without eithertranslocating from an enzyme binding site or dissociating from the APC;and (h) detecting and optionally quantifying the multiple abortiveoligonucleotide products.

Protein Detection

In another aspect of the invention, the methods disclosed herein may beused in diagnostic assays which detect proteins. As shown in FIG. 22, anabortive promoter cassette linker can be made with a protein modifiergroup attached, such that the linker is complementary to the APC linkerattached to the APC.

An illustrative procedure for detecting proteins, therefore, may includethe following process steps: (a) attaching a short piece of DNA of adefined sequence (APC linker) to a protein via a primary amine, asecondary amine, or a sulfhydral group; (b) retrieving and immobilizingthe modified protein with an antibody or some other affinity agentagainst the protein; and (c) attaching an abortive promoter cassette tothe protein by hybridization of the APC cassette to the APC linker onthe labeled protein; (d) detecting the protein by (i) treating the DNAwith an initiator nucleotide under hybridizing conditions; and (ii)treating the DNA with an RNA polymerase and nucleotides or nucleotideanalogs that permit detection. Process (d) occurs repeatedly for eachRNA polymerase bound.

Cancer Detection

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of cancer. Preferredmammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbitsand humans. Particularly preferred are humans.

The methods of the invention are particularly useful for monitoring thestatus of DNA methylation, genetic mutations, mRNA expression patterns,and protein expression patterns. The invention can be used to detect,diagnose, and monitor diseases, and/or disorders associated with theaberrant expression and/or activity of a polypeptide or polynucleotide.The invention provides for the detection of the aberrant expression of apolypeptide or polynucleotide, the presence of mutations, and changes inmethylation status of DNA. The method comprises (a) assaying theexpression of the polypeptide or polynucleotide of interest in cells,tissue or body fluid of an individual using the methods of abortiveinitiaton transcription described above, and (b) comparing the level ofgene expression, protein expression, or presence of sequences ofinterest with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide or polynucleotide level compared tothe standard level is indicative of aberrant expression indicatingpresence of cancer or a pathogen of interest.

The presence of an abnormal amount of transcript in biopsied tissue orbody fluid from an individual may indicate a predisposition for thedevelopment of cancer or a disease of interest, or may provide a meansfor detecting the disease prior to the appearance of actual clinicalsymptoms. A more definitive diagnosis of this type may allow healthprofessionals to employ preventative measures or aggressive treatmentearlier thereby preventing the development or further progression of thecancer or disease caused by the pathogen.

The diagnostic assays of the invention can be used for the diagnosis andprognosis of any disease, including but not limited to Alzheimerdisease, muscular dystrophy, cancer, breast cancer, colon cancer, cysticfibrosis, fragile X syndrome, hemophilia A and B, Kennedy disease,ovarian cancer, lung cancer, prostate cancer, retinoblastoma, myotonicdystrophy, Tay Sachs disease, Wilson disease, and Williams disease.These assays are believed to be particularly useful for the diagnosisand prognosis of all types of cancer.

Kits of the Invention

The invention also provides kits for carrying out the methods of theinvention. Such kits comprise, in one or more containers, usuallyconveniently packaged to facilitate their use in assays, quantities ofvarious compositions essential for carrying out the assays in accordancewith the invention. Thus, the kits comprise one or more initiatorsaccording to the invention. The kits may additionally comprise an enzymewith polymerase activity, such as an RNA and/or DNA polymerase forexample, to extend the primer of the kit, as well as reagents forprocessing a target nucleic acid. The kit may also comprise nucleotidesand/or nucleotide analogs to enable detection of the oligonucleotideproducts synthesized by the methods of the invention. The kits may alsoinclude oligonucleotide target site probes for forming a bubble complexon the target nucleic acid. The kit may also contain an abortivepromoter cassette. The kits may also contain components for thecollection and transport of materials, including but not limited to,membranes, affinity materials, test tubes, petri dishes, and dipsticks.The kit may also include microtiter plates, bio-chips, magnetic beads,gel matrices, or other forms of solid matrices to which anoligonucleotide capture probe, which is specific for a particular targetsequence, has been bound. The relative amounts of the components in thekits can be varied to provide for reagent concentrations thatsubstantially optimize the reactions involved in the practice of themethods disclosed herein and/or to further optimize the sensitivity ofany assay.

The test kits of the invention can also include, as is well-known tothose skilled in the art, various controls and standards, such assolutions of known target nucleic acid concentration, including notarget sequence (negative control), to ensure the reliability andaccuracy of the assays carried out using the kits and to permitquantitative analyses of test samples using the kits. Optionally, thekits may include a set of instructions, which are generally writteninstructions, though the instructions may be stored on electronicstorage media (e.g., magnetic diskette or optical disk), relating to theuse of the components of the methods of the invention. The instructionsprovided with the kit generally also include information regardingreagents (whether included or not in the kit) necessary or preferred forpracticing the methods of the invention, instructions on how to use thekit, and/or appropriate reaction conditions.

EXAMPLES

The following examples are provided for purposes of illustration onlyand not of limitation. Those of skill in the art will readily recognizea variety of non-critical parameters which could be changed or modifiedto yield essentially similar results.

Example 1 Synthesis of a Dye Labeled Initiator

One of several reactions to chemically modify a nucleotide is describedherein. 5′ a-S-CTP, which was purchased from TriLink BioTechnologies,was treated following the manufacturer's instructions with calfintestinal phosphatase. The phosphatase treatment is important becauseit increases the efficiency of the labeling reaction. Followingphosphatase treatment, 12.5 mM α-S-CMP was mixed with 5 μl of 0.2 MNaHCO3, 15 μl of DMF, and 15 μl of 90 mM IAEDANS (purchased fromMolecular Probes) in DMF, and incubated at room temperature for 1 hour.The reaction was extracted with 5 volumes water saturated ethyl ether.The aqueous phase was removed and the ether eliminated by evaporation.Thin layer chromatography was performed following standard protocolsknown in the art, and demonstrated that the reaction successfullyproduced 5′-IEADANS-S-CMP (FIG. 26).

Example 2 RNA Primer-Initiated Abortive Transcription with an RNAPolymerase

Reaction conditions have been optimized for abortive trancriptioninitiaton. The components and concentrations of Buffer T favor abortivetranscription initiation. Buffer T is comprised of: 20 mM Tris-HCl pH7.9, 5 mM MgCl₂, 5 mM beta-mercaptoethanol, 2.8% (v/v) glycerol. Primersare either ribonucleoside-triphosphates (NTPs) or dinucleotides rangingin concentration from 0.2-1.3 mM. Final NTP concentrations range from0.2-1.3 mM. The high ends of the concentration ranges are designed forpreparative abortive transcription. The template DNA concentration isless than 2 μM in terms of phosphate. E. coli RNA polymerase is added toa final concentration of between 15 nM and 400 nM. Either holoenzyme orcore can be used with a single-stranded template DNA. Yeast inorganicpyrophosphatase is added to 1 unit/ml in preparative reactions toprevent the accumulation of pyrophosphate. At high concentrationspyrophosphate can reverse the synthesis reaction causing RNA polymeraseto regenerate NTPs at the expense of the RNA products. One unit ofpyrophosphatase is defined as the amount of enzyme to liberate 1.0 μM ofinorganic orthophosphate per min. at 25° C. and pH 7.2. Reactions areincubated at 37° C. for up to 72 hours for preparative reactions. Theseconditions are representative; for specific templates, optimization ofparticular components and concentrations may enhance the efficiency ofabortive initiation.

Three different initiators were used in this example: (1) TAMARA-ApG;(2) Biotin ApG; and (3) ApG. The target nucleic acid template wasdenatured by boiling for 5 minutes at 95° C. and immediately placing onice. Each reaction was prepared as follows:

-   -   5.0 μl 1X Buffer T    -   2.5 μl of a-32P-UTP    -   14.3 μl ddH₂O    -   1 μl of E. coli RNA polymerase (1 U/μl)    -   100 ng (2 μl) of template DNA    -   10 nmoles (1.2 μl) of initiator    -   22.8 μl of reaction buffer

Incubate at 37° C. for 12-16 hours. Thin layer chromatography wasperformed using standard methods known in the art to determine theextent of incorporation of UTP in the third position (FIG. 27).

Both TAMARA-ApG and biotin ApG allowed for incorporation of thenucleotide UTP. Biotin ApG incorporated more efficiently thanTAMARA-ApG, but not as efficient as ApG.

Example 3 Abortive Initiation Reaction with a Labeled Terminator

Abortive transcription initiation reactions may be performed with alabeled initiator and/or a labeled terminator. The following reactionconditions were used to incorporate a labeled terminator:

-   -   5 μl 1X Buffer T    -   3 μl 100 ng denatured DNA template (pBR322)    -   13.5 μl dd H₂O    -   1 μl E. coli RNA polymerase    -   1.2 μl dinucleotide initiator ApG    -   1.5 μl of 7 mM SF-UTP

Incubate mixture at 37° C. for 16 hours in temperature controlledmicrotitre plate reader. Thin layer chromatography was performed usingstandard methods known in the art, and demonstrated that the labeledtrinucleotide ApGpU was generated (FIG. 28).

Example 4 Fluorescent Energy Transfer Between Donors and Acceptors

The above examples have demonstrated that both labeled initiators andterminators can be incorporated into the oligonucleotide products. Oneefficient method to measure incorporation of the labeled nucleotides isby Fluorescent Resonance Energy Transfer. The following conditions wereused to demonstrate FRET between a labeled initiator and a labeledterminator:

-   -   5 μl 1X Reaction Buffer (Buffer T)    -   3 μl denatured DNA template (300 ng pBR322)    -   13.5 μl dd H₂O    -   1 μl E. coli RNA polymerase    -   1.2 μl Initiator (TAMARA-ApG or ApG or Biotin-ApG)    -   1.5 μl of of 7 mM SF-UTP

The reaction mixture was incubated at 37C for 16 hours in temperaturecontrolled microtitre plate reader, which was set to read at thefollowing parameters: Ex 485, Em 620, Gain 35, 99 reads/well/cycle.Under the reaction conditions described above, the RNA polymerasereiteratively synthesizes an oligonucleotide product composed of theinitiator (TAMARA-SpApG) and the terminator (SF-UTP).

Formation of the oligonucleotide product, TAMARA-SpApGpU-SF, places theinitiator and the terminator within 80 angstroms of each other, whichallows for the transfer of energy between the chemical moieties. Energyis transferred from the donor, which is SF-UTP, to the acceptor, whichis TAMARA-ApG. This transfer of energy can be detected and/orquantitated by a change in wavelength emission of TAMARA (TAMARAAbosrbance=540 nm; Emission=565 nm)

As the oligonucleotide product is generated, energy transfer occursbetween TAMARA-SpApG and SF-UTP, which changes the wavelength at whichTAMARA emits. If RNA polymerase or DNA is omitted from the reaction,there is no transfer of energy between the initiator and the terminator,and no change in the wavelength at which TAMARA emits.

Example 5 Determination of the Methylation Status of Specific Residuesof the CDKN2A Gene

The sample to be analyzed is collected from a human stool sample.Methods of DNA extraction from stool samples are well known in the art,and commercial kits are avialalbe for extracting human DNA from stoolsamples, such as QIAamp DNA Stool Mini Kit from Qiagen (Valencia,Calif.).

After extraction, the sample is applied to the wells of a microtiterplate, which contain a capture probe for the gene of interest, in thisparticular example, the capture probe is for CDKN2A gene. The nucleotidesequence of a representative capture probe for the CpG islands of theCDKN2A gene is as follows:

ATATACTGGGTCTACAAGGTTTAAGTCAACCAGGGATTGAAATATAACTTTT AAACAGAGCTGG. (SEQID NO: 16). The DNA sample is incubated with the capture probe to allowhybridization. A representative hybridization protocol is as follows:(1) prehybridize with 2.5×SSC, 5× Denhardt's at room temperature for 30minutes; (2) hybridize with 2.5×SSC, 5× Denhardt's, 30% formamide atroom temperature for 2 hours; (3) wash twice with 1×SSC at 42° C. for 10minutes, maintaining 42° C.; and (4) wash three times with 0.1×SSC at42° C. for 10 minutes, maintaining 42° C.

The DNA is treated with a deaminating agent, such as sodium bisulfite,which will de-aminate the unmethylated C's in the DNA, while leaving themethylated C's unaltered. The wells are then washed under mediumstringency conditions to remove the remaining sodium bisulfite.

A representative transcription reaction is comprised of the followingcomponents: E. coli holoenzyme RNA polymerase; reaction buffer: 10 nMTris-HCl, pH 7.0; 10 mM KCl; 0.5 mM Na₂EDTA; and 50 mg/ml BSA; aninitiator, and nucleotide analogs. The reaction conditions forparticular nucleotide sequence may vary. Other polymerases may be used,such as E. coli T7, or SP6. The reaction buffer can be optimized toincrease abortive initiation events by adjusting the salt concentration,divalent cations and concentrations, the glycerol content, and theamount and type of reducing agent to be used.

The initiator will be a 5′-αSpCpG dimer labeled through the 5′-S withfluorescein, which fucntions as the donor in this reaction. Thenucleotide analog(s) will be labeled with TAMARA, which will function asthe acceptor in this reaction. The initiator can be labeled with eitherthe donor or the acceptor in the FRET reaction, and dependending uponthe fluorescent molecule used to label the initiator, the nucleotideanalog(s) will be labeled with either a donor or an acceptor.

Fluorescein is excited using a 360 nm wavelength filter; the resultingemission peak is at about 515 nm. If the TAMARA is in close proximity tothe fluorescein, it becomes excited at 542 nm, resulting in an emissionpeak of 568 nm. The near ultraviolet wavelength excties the fluoresceinbut not the rhodamine. Therefore signal will only be generated if thefluorescein is in close proximity to the rhodamine. This signal can begenerated and monitored in a fluorescent microtitre plate reader thathas been fitted with specific excitation and emission filters for thisFRET pair. These filters and plate readers are commercially availablefrom a number of sources, although most clinical labs and researchfacilities already use a fluorescent microtitre plate reader.

In the foregoing specification, the invention has been described withreference to specific embodiments. However, it will be appreciated thatvarious modifications and changes can be made without departing from thescope of the present invention as set forth in the claims below. Thespecification and figures are to be regarded in an illustrative manner,rather than a restrictive one, and all such modifications are intendedto be included within the scope of present invention. Accordingly, thescope of the invention should be determined by the appended claims andtheir legal equivalents, rather than by the examples given above. Forexample, the steps recited in any of the method or process claims may beexecuted in any order and are not limited to the order presented in theclaims.

1. A method for detecting a single stranded DNA or RNA targetpolynucleotide in a test sample, said method comprising: (a) hybridizingsaid single stranded target polynucleotide with a target-specific linkerof an abortive promoter cassette, wherein said abortive promotercassette comprises said target-specific linker that hybridizes with saidsingle-stranded target polynucleotide wherein said target-specificlinker is a nucleic acid and comprises a single-stranded region of 5 to40 nucleotides that is an overhang region of said cassette, and, at the5′ or 3′ end of said target specific linker, a self-complementary DNAsequence and an RNA-polymerase binding site, wherein said selfcomplementary sequence comprises two partially complementary upper andlower oligonucleotides that form a single-stranded transcription bubbleregion comprising a defined site from which an initiator and a suitableRNA polymerase can synthesize an abortive oligonucleotide product; (b)incubating said hybridized target polynucleotide and linker of part (a)with an RNA polymerase, an initiator, and a terminator; (c) synthesizingan oligonucleotide transcript that is complementary to the initiationstart site of said abortive promoter cassette by an abortive,reiterative process, wherein said process does not use saidsingle-stranded target polynucleotide as a template and wherein saidinitiator is extended until said terminator is incorporated into saidoligonucleotide transcript, thereby synthesizing multiple abortivereiterative oligonucleotide transcripts; and (d) detecting orquantifying said reiterative oligonucleotide transcripts.
 2. A methodfor detecting the presence of a pathogen in a test sample, said methodcomprising: (a) hybridizing a single stranded target pathogenpolynucleotide in said test sample with a target-specific linker of anabortive promoter cassette, wherein said abortive promoter cassettecomprises said target-specific linker that hybridizes with saidsingle-stranded target pathogen polynucleotide wherein saidtarget-specific linker is a nucleic acid and comprises a single-strandedregion of 5 to 40 nucleotides that is an overhang region of saidcassette, and, at the 5′ or 3′ end of said target specific linker, aself-complementary DNA sequence and an RNA-polymerase binding site,wherein said self complementary sequence comprises two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; (b) incubating said hybridized targetpolynucleotide and linker of part (a) with an initiator an RNApolymerase, and a terminator; (c) synthesizing an oligonucleotidetranscript that is complementary to the initiation start site of theabortive promoter cassette by an abortive, reiterative process, whereinsaid process does not use said single-stranded target pathogenpolynucleotide as a template and wherein said initiator is extendeduntil said terminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (d) determining the presence of said pathogen by detecting orquantifying said reiterative oligonucleotide transcripts synthesized insaid test sample
 3. The method of any one of claims 1 or 2, furthercomprising detecting or quantifying said reiteratively synthesizedoligonucleotide transcript by modifying a nucleotide in at least one ofthe members selected from the group consisting of said terminator, andsaid initiator.
 4. The method of claim 3, wherein said modifyingcomprises incorporating a label moiety.
 5. The method of claim 4,wherein said label moiety comprises a fluorophore moiety.
 6. The methodof claim 5, wherein said fluorophore moiety comprises a fluorescentenergy donor and a fluorescent energy acceptor wherein said moiety isdetected or quantified by fluorescence resonance energy transfer.
 7. Themethod of claim 5 wherein said fluorophore moiety is selected from thegroup consisting of:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate; 5-(2′-aminoethyl)anininonaphthalene-1 -sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl) phenyl]naphthalimide-3 ,5 disulfonate;N-(4-amino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin, and derivatives: coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate; erythrosin and derivatives:erytbrosin B, erytbrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1pyrene; butyrate quantum dots; ReactiveRed 4; rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B, sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbiun chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800;La Jolla Blue; phihalo cyanine; and naphthalo cyanine.
 8. The method ofany one of claims 1 or 2, wherein said polymerase is selected from thegroup consisting of: a DNA-dependent RNA polymerase, an RNA-dependentRNA polymerase and a modified RNA polymerase, and a primase.
 9. Themethod of claim 8, wherein said polymerase comprises an RNA polymerasederived from one of E. coli, E. coli bacteriophage T7, E. colibacteriophage T3, and S. typhimurium bacteriophage SP6.
 10. The methodof any one of claims 1 or 2, wherein said abortive oligonucleotides thatare synthesized are 2 to 100 nucleotides long.
 11. The method of any oneof claims 1 or 2, wherein said terminator comprises a nucleotide analog.12. The method of claim 1 or 1, wherein said initiator comprisesnucleotides selected from the group consisting of: 1-25 nucleotides,26-50 nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125nucleotides, and 126-150 nucleotides, 151-175 nucleotides, 176-200nucleotides, 201-225 nucleotides, 226-250 nucleotides, and greater than250 nucleotides.
 13. The method of claim 12, wherein said initiator is1-250 nucleotides long.
 14. The method of claim 2, wherein saidsingle-stranded target pathogen polynucleotide is DNA or RNA.
 15. Themethod of any one of claims 1 or 2, wherein said inititator is RNA. 16.A method for detecting a pathogen in a test sample, said methodcomprising: (a) immobilizing a capture probe designed to hybridize witha single stranded target pathogen polynucleotide in said test sample;(b) hybridizing said capture probe with a test sample that potentiallycontains said single stranded target pathogen polynucleotide; (c)hybridizing said target polynucleotide in said test sample in adifferent region than the hybridization region mentioned in (b) with atarget-specific linker of an abortive promoter cassette, wherein saidabortive promoter cassette comprises said target-specific linker thathybridizes with said single-stranded target pathogen polynucleotidewherein said target-specific linker is a nucleic acid and comprises asingle-stranded region of 5 to 40 nucleotides that is an overhang regionof said cassette, and, at the 5′ or 3′ end of said target specificlinker, a self-complementary DNA sequence and an RNA-polymerase bindingsite, wherein said self complementary seciuence comprises two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; (d) incubating the hybridized targetpathogen polynucleotide and linker of part (c) with an RNA-polymerase,initiator, and a terminator; (e) synthesizing an oligonucleotidetranscript that is complementary to the transcription initiation startsite of said abortive promoter cassette by an abortive, reiterativeprocess, wherein said process does not use said single-stranded targetpathogen polynucleotide as template and wherein said initiator isextended until said terminator is incorporated into saidoligonucleotides thereby synthesizing multiple abortive reiterativeoligonucleotide transcripts; and (f) determining the presence or absenceof said pathogen by detecting or quantifying said reiterativeoligonucleotide transcripts.
 17. A method for detecting mRNA expressionin a test sample, the method comprising: (a) hybridizing a target mRNAsequence with a target specific linker of an abortive promoter cassette,wherein said abortive promoter cassette comprises said target-specificlinker that hybridizes with said target mRNA sequence wherein saidtarget-specific linker is a nucleic acid and comprises a single-strandedregion of 5 to 40 nucleotides that is an overhang region of saidcassette, and, at the 5′ or 3′ end of said target specific linker, aself-complementary DNA sequence and an RNA-polymerase binding site,wherein said self complementary sequence comprises two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; (b) incubating said hybridized targetmRNA sequence and linker of part (a) with an RNA-polymerase, aninitiator, and a terminator; (c) synthesizing an oligonucleotidetranscript that is complementary to the transcription initiation startsite by an abortive, reiterative process, wherein said process does notuse said target mRNA as a template and wherein said initiator isextended until said terminator is incorporated into said oligonucleotidetranscript, thereby synthesizing multiple reiterative oligonucleotides;and (d) determining the presence or absence of said mRNA by detecting orquantifying said reiterative oligonucleotide transcripts.
 18. The methodof claim 17, further comprising: (a) immobilizing a capture probe,wherein said probe hybridizes with a target mRNA sequence in a differentregion than the hybridization region of a target specific linker of anabortive promoter cassette; (b) hybridizing said capture probe with atest sample which potentially contains said target mRNA sequence; and(c) washing a captured target mRNA sequence to remove unhybridizedcomponents of said test sample.
 19. The method of claim 17, furthercomprising detecting or quantifying said reiteratively synthesizedoligonucleotide transcript by modifying a nucleotide in at least one ofthe members selected from the group consisting of said terminator, saidinitiator, wherein said modifying comprises incorporating anindependently selected label moiety into at least one of said initiator,and said terminator.
 20. The method of claim 19, wherein said labelmoiety comprises a fluorophore moiety.
 21. The method of claim 20,wherein detecting comprises detecting by fluorescence resonance energytransfer and said fluorophore moiety comprises one of a fluorescentenergy donor and a fluorescent energy acceptor.
 22. The method of claim17, wherein said polymerase is one of a DNA-dependent RNA polymerase, anRNA-dependent RNA polymerase, an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, and a modified polymerase, and a primase.23. The method of claim 17, wherein said polymerase comprises an RNApolymerase derived from one of E. coli, E. coli bacteriophage T7, E.coli bacteriophage T3, and S. typhimurium bacteriophage SP6.
 24. Themethod of claim 17, wherein said initiator is one of RNA or DNA.
 25. Themethod of claim 24, wherein said initiator comprises nucleotidesselected from the group consisting of: 1-25 nucleotides, 26-50nucleotides, 51-75 nucleotides, 76-100 nucleotides, 101-125 nucleotides,126-150 nucleotides, 151-175 nucleotides, 176-200 nucleotides, 201-225nucleotides, 226-250 nucleotides, and greater than 250 nucleotides. 26.The method of claim 17, wherein said abortive oligonucleotides beingsynthesized are one of the lengths selected from the group consistingof: about 2 to about 26 nucleotides, about 26 to about 50 nucleotides,about 50 nucleotides to about 100 nucleotides, and greater than 100nucleotides.
 27. The method of claim 17, wherein said target-specificlinker of an abortive promoter cassette hybridizes with a poly-A tail ofsaid target mRNA sequence.
 28. The method of claim 17, wherein saidchain terminator is a nucleotide analog.
 29. A method for detecting atarget protein in a test sample, the method comprising: (a) covalentlyattaching said target protein to a reactive target specific linker of anabortive promoter cassette, wherein said abortive promoter cassettecomprises (i) a self-complementary DNA sequence and an RNA-polymerasebinding site: wherein said self complementary sequence is selected fromthe group consisting of: a) one contiguous oligonucleotide to which RNApolymerase can bind to form a transcription bubble; b) two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; and c) two complementaryoligonucleotides that form a transcription bubble region in the presenceof an RNA polymerase, which allows for the synthesis of an abortiveoligonucleotide product; and (ii) said target-specific linker on atleast the 3′ or 5′ end of one strand with the proviso that when thetarget specific linker is a nucleic acid, the linker comprises asingle-stranded overhang region of 5 to 40 nucleotides with the furtherproviso that when the abortive promoter cassette is that of part (a)(i)or (a)(iii), the target specific linker is not a nucleic acid; (b)incubating said abortive promoter cassette that has said covalentlyattached target protein with an RNA-polymerase, an initiator, and aterminator; (c) synthesizing an oligonucleotide transcript that iscomplementary to the transcription initiation start site of the abortivepromoter cassette, wherein said initiator is extended until saidterminator is incorporated into said oligonucleotide transcript, therebysynthesizing multiple reiterative oligonucleotide transcripts; and (d)determining the presence or absence of the target protein by detectingor quantifying said reiterative oligonucleotide transcripts.
 30. Themethod of claim 29 further comprising immobilizing target protein by atarget specific probe.
 31. The method of claim 30, wherein said targetspecific probe is an antibody.
 32. The method of claim 29, wherein saidtarget specific linker of an abortive promoter cassette is covalentlyattached to the target protein by thiol-reactive or amine-reactivecrosslinking agents.
 33. The method of claim 32 wherein said proteincrosslinking agents are selected from the group consisting of:maleamides, iodoacetamides, and disulfides.
 34. The method of claim 29,wherein said target protein is purified or in a cell lysate.
 35. Amethod for detecting a pathogen, said method comprising: (a) obtaining asample in need of detection of said pathogen (b) hybridizing a singlestranded target pathogen polynucleotide in said sample with a targetspecific linker of an abortive promoter cassette, wherein said abortivepromoter cassette comprises said target-specific linker that hybridizeswith said single-stranded target pathogen polynucleotide wherein saidtarget-specific linker is a nucleic acid and comprises a single-strandedregion of 5 to 40 nucleotides that is an overhang region of saidcassette, and, at the 5′ or 3′ end of said target specific linker, aself-complementary DNA sequence and an RNA-polymerase binding site,wherein said self complementary seciuence comprises two partiallycomplementary upper and lower oligonucleotides that form asingle-stranded transcription bubble region comprising a defined sitefrom which an initiator and a suitable RNA polymerase can synthesize anabortive oligonucleotide product; (c) incubating the hybridized targetpathogen polynucleotide and linker of part (a) with an initiator an RNApolymerase, and a terminator; (d) synthesizing an oligonucleotidetranscript that is complementary to the initiation start site of theabortive promoter cassette by an abortive, reiterative process, whereinsaid process does not use said single-stranded target pathogenpolynucleotide as template and said initiator is extended until saidterminator is incorporated into said oligonucleotides therebysynthesizing multiple abortive reiterative oligonucleotide transcripts;and (e) determining the presence of said pathogen by detecting orquantifying said reiteratively synthesized oligonucleotide transcriptssynthesized said sample.
 36. The method of claim 35, wherein said methodfurther comprises: immobilizing an oligonucleotide capture probe whichis specific for said target pathogen polynucleotide; and hybridizingsaid oligonucleotide capture probe with a denatured DNA sample whichpotentially contains said target pathogen polynucleotide in a differentregion than the hybridization region of a target specific linker of anabortive promoter cassette.
 37. The method of claim 35, wherein saidsample is obtained from the group consisting of: animal, plant or humantissue, blood, saliva, semen, urine, sera, cerebral or spinal fluid,pleural fluid, lymph, sputum, fluid from breast lavage, mucusoalsecretions, animal solids, stool, cultures of microorganisms, liquid andsolid food and feed-products, waste, cosmetics, air and water.
 38. Themethod of any one of claims 1, 2, 16 or 35, wherein said initiator isselected from the group consisting of: nucleosides, nucleoside analogs,nucleotides, and nucleotide analogs.
 39. The method of any one of claims1 2, 16 or 35, further comprising incubating said target polynucleotidewith additional ribonucleotides.
 40. The method of claim 39, whereinsaid ribonucleotides are modified.
 41. The method of claim 40, whereinsaid modification comprises incorporating a labeling moiety.
 42. Amethod for detecting a single stranded DNA or RNA target polynucleotidein a test sample, said method comprising: (a) hybridizing said singlestranded target polynucleotide with a target-specific linker of anabortive promoter cassette, wherein said abortive promoter cassettecomprises said target-specific linker that hybridizes with saidsingle-stranded target polynucleotide wherein said target-specificlinker is a nucleic acid and comprises a single-stranded region of 5 to40 nucleotides that is an overhang region of said cassette, and, at the5′ or 3′ end of said target specific linker, a self-complementary DNAsequence and an RNA-polymerase binding site, wherein said selfcomplementary sequence comprises two partially complementary upper andlower oligonucleotides that form a single-stranded transcription bubbleregion comprising a defined site from which an initiator and a suitableRNA polymerase can synthesize an abortive oligonucleotide product; (b)incubating said hybridized target polynucleotide and linker of part (a)with an RNA polymerase and an initiator; (c) synthesizing anoligonucleotide transcript that is complementary to the initiation startsite of said abortive promoter cassette by an abortive, reiterativeprocess, wherein said process does not use said single-stranded targetpolynucleotide as a template and wherein said initiator is extendeduntil termination occurs through nucleotide deprivation; therebysynthesizing multiple reiterative oligonucleotide transcripts; and (e)detecting or quantifying said reiterative oligonucleotide transcripts.43. The method of claim 42 further comprising: (a) immobilizing acapture probe designed to hybridize with a target polynucleotide in saidtest sample; (b) hybridizing said capture probe with a test sample thatpotentially contains said target polynucleotide in a different regionthan the hybridization region of a target specific linker of an abortivepromoter cassette.
 44. The method of claims 17, 29 or 42, wherein saidmethod detects the presence of a pathogen in a test sample.
 45. Themethod of any one of claims 2, 16, 17, 29 35 or 42, wherein the presenceof a virus is detected.
 46. The method of any one of claims 2, 16, 17,29 35 or 42 wherein the presence of a bacteria is detected.
 47. Themethod of any one of claims 1, 2, 16,17 35 or 42 wherein the targetpolynucleotide is RNA.
 48. The method of claim 47, wherein the RNA ismRNA.
 49. The method of claim 47, wherein the RNA polymerase is anRNA-dependent RNA polymerase.
 50. The method of claim 49 wherein theRNA-dependent RNA-polymerase is poliovirus RNA polymerase.
 51. Themethod of claim 47, further comprising incubating said RNA with areverse transcriptase enzyme.
 52. The method of any one of claims 1, 2,16, 17, 29, 35 or 42, wherein said initiator is 1-3 bases in length.